RSV-pos

[{"id":42,"details":{"paperId":"c5983be79e5dae3ce8865febd927d70b7f557c57","externalIds":{"MAG":"2061680252","DOI":"10.1007/s12104-011-9308-3","CorpusId":"22182180","PubMed":"21523439"},"title":"1H, 13C, and 15N resonance assignment of the central domain of hRSV transcription antitermination factor M2-1","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Almost complete 1H, 13C and 15N assignment of a fragment of M2-1 corresponding to a central domain located between residues Glu59 and Lys177 is reported, for further structure determination and interaction studies."}},"tag":"EXP"},{"id":292,"details":{"paperId":"aba75c3f74252b3ff61227f6e27a66f1da438f7a","externalIds":{"MAG":"1785948735","DOI":"10.1128/JVI.01076-15","CorpusId":"2557468","PubMed":"26178980"},"title":"2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1","abstract":"ABSTRACT 2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is concluded that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth."}},"tag":"EXP"},{"id":415,"details":{"paperId":"184448a5408a638d16ce11da120943c4979a3ee5","externalIds":{"MAG":"1195517654","DOI":"10.1159/000435774","CorpusId":"2303252","PubMed":"26305094"},"title":"4EBP1 Is Dephosphorylated by Respiratory Syncytial Virus Infection","abstract":"Respiratory syncytial virus (RSV) requires protein biosynthesis machinery to generate progeny. There is evidence that RSV might alter some translation components since stress granules are formed in their host cells. Consistent with these observations, we found that RSV induces dephosphorylation of 4EBP1 (eIF4E-binding protein), an important cellular translation factor. Our results show no correlation between the 4EBP1 dephosphorylation time and the decrease in the global rate of protein synthesis. Interestingly, treatment with rapamycin stimulates virus generation. The results suggest that RSV is a virus that still contains unknown mechanisms involved in the translation of their mRNAs through the alteration or modification of some translation factors, such as 4EBP1, possibly to favor its replicative cycle.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results suggest that RSV is a virus that still contains unknown mechanisms involved in the translation of their mRNAs through the alteration or modification of some translation factors, such as 4EBP1, possibly to favor its replicative cycle."}},"tag":"EXP"},{"id":471,"details":{"paperId":"b43263fe4ce07428cde942761f3826a95177f704","externalIds":{"MAG":"3163449344","DOI":"10.21203/RS.3.RS-515369/V1","CorpusId":"236385831"},"title":"8-oxoguanine DNA glycosylase1 recognizes oxidatively-generated epitranscriptomic marks on nascent mRNAs to promote RSV replication","abstract":"\n Respiratory syncytial virus (RSV) infection induces an oxidizing environment linked to increased viral load, expression of pro-inflammatory genes, and excessive lung inflammation. The mechanisms of how reactive oxygen species (ROS) promotes viral gene expression have remained largely elusive. Here we show that nascent (n)RNAs of RSV acquire 8-oxo-7,8-dihydroguanine (8-oxo(r)Gua) -a covalently modified guanine base in their 5’-UTR peritranscriptionally, while paired with the 3’-terminus of viral gene(s). 8-oxo(r)Gua is bound by 8-oxoguanine DNA glycosylase1 (OGG1), a complex that physically interacts with and recruits the anti-terminator protein M2-1 to increase viral gene transcription. Knockdown of OGG1 (but not other DNA glycosylases) or inhibition of its binding, significantly decreased RSV mRNA, protein levels and yield of progeny in cultured cells and airways. Collectively, these data suggest that Gua oxidation in vRNA, serves as an epitranscriptomic mark that repurposes OGG1 to increase lytic viral replication. Pharmacological inhibition of OGG1 binding to the epitranscriptomic mark could have clinical utility to decrease manifestations of RSV infection.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Data suggest that Gua oxidation in vRNA, serves as an epitranscriptomic mark that repurposes OGG1 to increase lytic viral replication, which could have clinical utility to decrease manifestations of RSV infection."}},"tag":"EXP"},{"id":396,"details":{"paperId":"096bf8e7405369aad1592e24a123155a80e9e6c3","externalIds":{"MAG":"2056379037","PubMedCentral":"3280462","DOI":"10.1128/mBio.00270-11","CorpusId":"7268247","PubMed":"22318318"},"title":"A Critical Phenylalanine Residue in the Respiratory Syncytial Virus Fusion Protein Cytoplasmic Tail Mediates Assembly of Internal Viral Proteins into Viral Filaments and Particles","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is a single-stranded RNA virus in the Paramyxoviridae family that assembles into filamentous structures at the apical surface of polarized epithelial cells. These filaments contain viral genomic RNA and structural proteins, including the fusion (F) protein, matrix (M) protein, nucleoprotein (N), and phosphoprotein (P), while excluding F-actin. It is known that the F protein cytoplasmic tail (FCT) is necessary for filament formation, but the mechanism by which the FCT mediates assembly into filaments is not clear. We hypothesized that the FCT is necessary for interactions with other viral proteins in order to form filaments. In order to test this idea, we expressed the F protein with cytoplasmic tail (CT) truncations or specific point mutations and determined the abilities of these variant F proteins to form filaments independent of viral infection when coexpressed with M, N, and P. Deletion of the terminal three FCT residues (amino acids Phe-Ser-Asn) or mutation of the Phe residue resulted in a loss of filament formation but did not affect F-protein expression or trafficking to the cell surface. Filament formation could be restored by addition of residues Phe-Ser-Asn to an FCT deletion mutant and was unaffected by mutations to Ser or Asn residues. Second, deletion of residues Phe-Ser-Asn or mutation of the Phe residue resulted in a loss of M, N, and P incorporation into virus-like particles. These data suggest that a C-terminal Phe residue in the FCT mediates assembly through incorporation of internal virion proteins into virus filaments at the cell surface. IMPORTANCE Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide. There is no licensed RSV vaccine and only limited therapeutics for use in infected patients. Many aspects of the RSV life cycle have been studied, but the mechanisms that drive RSV assembly at the cell surface are not well understood. This study provides evidence that a specific residue in the RSV fusion protein cytoplasmic tail coordinates assembly into viral filaments by mediating the incorporation of internal virion proteins. Understanding the mechanisms that drive RSV assembly could lead to targeted development of novel antiviral drugs. Moreover, since RSV exits infected cells in an ESCRT (endosomal sorting complexes required for transport)-independent manner, these studies may contribute new knowledge about a general strategy by which ESCRT-independent viruses mediate outward bud formation using viral protein-mediated mechanisms during assembly and budding. Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide. There is no licensed RSV vaccine and only limited therapeutics for use in infected patients. Many aspects of the RSV life cycle have been studied, but the mechanisms that drive RSV assembly at the cell surface are not well understood. This study provides evidence that a specific residue in the RSV fusion protein cytoplasmic tail coordinates assembly into viral filaments by mediating the incorporation of internal virion proteins. Understanding the mechanisms that drive RSV assembly could lead to targeted development of novel antiviral drugs. Moreover, since RSV exits infected cells in an ESCRT (endosomal sorting complexes required for transport)-independent manner, these studies may contribute new knowledge about a general strategy by which ESCRT-independent viruses mediate outward bud formation using viral protein-mediated mechanisms during assembly and budding.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Evidence is provided that a specific residue in the RSV fusion protein cytoplasmic tail coordinates assembly into viral filaments by mediating the incorporation of internal virion proteins into virus filaments at the cell surface."}},"tag":"EXP"},{"id":403,"details":{"paperId":"868625d0d912b4fe394414722bfc851b5829f849","externalIds":{"MAG":"2461688869","PubMedCentral":"5069771","DOI":"10.1128/mSystems.00051-16","CorpusId":"2684993","PubMed":"27822537"},"title":"A Simple Screening Approach To Prioritize Genes for Functional Analysis Identifies a Role for Interferon Regulatory Factor 7 in the Control of Respiratory Syncytial Virus Disease","abstract":"Making the most of “big data” is one of the core challenges of current biology. There is a large array of heterogeneous data sets of host gene responses to infection, but these data sets do not inform us about gene function and require specialized skill sets and training for their utilization. Here we describe an approach that combines and simplifies these data sets, distilling this information into a single list of genes commonly upregulated in response to infection with RSV as a model pathogen. Many of the genes on the list have unknown functions in RSV disease. We validated the gene list with new clinical, in vitro, and in vivo data. This approach allows the rapid selection of genes of interest for further, more-detailed studies, thus reducing time and costs. Furthermore, the approach is simple to use and widely applicable to a range of diseases. ABSTRACT Greater understanding of the functions of host gene products in response to infection is required. While many of these genes enable pathogen clearance, some enhance pathogen growth or contribute to disease symptoms. Many studies have profiled transcriptomic and proteomic responses to infection, generating large data sets, but selecting targets for further study is challenging. Here we propose a novel data-mining approach combining multiple heterogeneous data sets to prioritize genes for further study by using respiratory syncytial virus (RSV) infection as a model pathogen with a significant health care impact. The assumption was that the more frequently a gene is detected across multiple studies, the more important its role is. A literature search was performed to find data sets of genes and proteins that change after RSV infection. The data sets were standardized, collated into a single database, and then panned to determine which genes occurred in multiple data sets, generating a candidate gene list. This candidate gene list was validated by using both a clinical cohort and in vitro screening. We identified several genes that were frequently expressed following RSV infection with no assigned function in RSV control, including IFI27, IFIT3, IFI44L, GBP1, OAS3, IFI44, and IRF7. Drilling down into the function of these genes, we demonstrate a role in disease for the gene for interferon regulatory factor 7, which was highly ranked on the list, but not for IRF1, which was not. Thus, we have developed and validated an approach for collating published data sets into a manageable list of candidates, identifying novel targets for future analysis. IMPORTANCE Making the most of “big data” is one of the core challenges of current biology. There is a large array of heterogeneous data sets of host gene responses to infection, but these data sets do not inform us about gene function and require specialized skill sets and training for their utilization. Here we describe an approach that combines and simplifies these data sets, distilling this information into a single list of genes commonly upregulated in response to infection with RSV as a model pathogen. Many of the genes on the list have unknown functions in RSV disease. We validated the gene list with new clinical, in vitro, and in vivo data. This approach allows the rapid selection of genes of interest for further, more-detailed studies, thus reducing time and costs. Furthermore, the approach is simple to use and widely applicable to a range of diseases.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A novel data-mining approach combining multiple heterogeneous data sets to prioritize genes for further study by using respiratory syncytial virus (RSV) infection as a model pathogen with a significant health care impact is proposed."}},"tag":"EXP"},{"id":486,"details":{"paperId":"b7283f1832c0076eaf92a5553672b5c55c0ba221","externalIds":{"PubMedCentral":"8392014","DOI":"10.3390/biom11081225","CorpusId":"237321690","PubMed":"34439894"},"title":"A Structural and Dynamic Analysis of the Partially Disordered Polymerase-Binding Domain in RSV Phosphoprotein","abstract":"The phosphoprotein P of Mononegavirales (MNV) is an essential co-factor of the viral RNA polymerase L. Its prime function is to recruit L to the ribonucleocapsid composed of the viral genome encapsidated by the nucleoprotein N. MNV phosphoproteins often contain a high degree of disorder. In Pneumoviridae phosphoproteins, the only domain with well-defined structure is a small oligomerization domain (POD). We previously characterized the differential disorder in respiratory syncytial virus (RSV) phosphoprotein by NMR. We showed that outside of RSV POD, the intrinsically disordered N-and C-terminal regions displayed a structural and dynamic diversity ranging from random coil to high helical propensity. Here we provide additional insight into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD. By using small phosphoprotein fragments centered on or adjacent to POD, we obtained a structural picture of the POD–PCα region in solution, at the single residue level by NMR and at lower resolution by complementary biophysical methods. We probed POD–PCα inter-domain contacts and showed that small molecules were able to modify the dynamics of PCα. These structural properties are fundamental to the peculiar binding mode of RSV phosphoprotein to L, where each of the four protomers binds to L in a different way.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Insight is provided into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD, and POD–PCα inter-domain contacts are probed and it is shown that small molecules were able to modify the dynamics of PC α."}},"tag":"EXP"},{"id":149,"details":{"paperId":"47a1ff9f556d2d7412476cd4479ba3ec34048995","externalIds":{"MAG":"2612310777","DOI":"10.1039/c7nr02162c","CorpusId":"206099890","PubMed":"28561831"},"title":"A dynamic cell entry pathway of respiratory syncytial virus revealed by tracking the quantum dot-labeled single virus.","abstract":"Studying the cell entry pathway at the single-particle level can provide detailed and quantitative information for the dynamic events involved in virus entry. Indeed, the viral entry dynamics cannot be monitored by static staining methods used in cell biology, and thus virus dynamic tracking could be useful in the development of effective antiviral strategies. Therefore, the aim of this work was to use a quantum dot-based single-particle tracking approach to monitor the cell entry behavior of the respiratory syncytial virus (RSV) in living cells. The time-lapse fluorescence imaging and trajectory analysis of the quantum dot-labeled RSV showed that RSV entry into HEp-2 cells consisted of a typical endocytosis trafficking process. Three critical events during RSV entry were observed according to entry dynamic and fluorescence colocalization analysis. Firstly, RSV was attached to lipid rafts of the cell membrane, and then it was efficiently delivered into the perinuclear region within 2 h post-infection, mostly moving and residing into the lysosome compartment. Moreover, the relatively slow velocity of RSV transport across the cytoplasm and the formation of the actin tail indicated actin-based RSV motility, which was also confirmed by the effects of cytoskeletal inhibitors. Taken together, these findings provided new insights into the RSV entry mechanism and virus-cell interactions in RSV infection that could be beneficial in the development of antiviral drugs and vaccines.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The aim of this work was to use a quantum dot-based single-particle tracking approach to monitor the cell entry behavior of the respiratory syncytial virus (RSV) in living cells and provide new insights into the RSV entry mechanism and virus-cell interactions in RSV infection."}},"tag":"EXP"},{"id":53,"details":{"paperId":"2d0a16b6e814653b8a8f0fa3e6f79c6023e0cb4b","externalIds":{"DOI":"10.1016/j.abb.2022.109424","CorpusId":"252769314","PubMed":"36220378"},"title":"A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles is uncovered, uncovering diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid."}},"tag":"EXP"},{"id":187,"details":{"paperId":"f8c5e5723659bff0d839264073f7949b6fb26ce5","externalIds":{"PubMedCentral":"5501218","MAG":"2613527233","DOI":"10.1080/19420889.2017.1319025","CorpusId":"11007517","PubMed":"28702128"},"title":"A novel host factor for human respiratory syncytial virus","abstract":"ABSTRACT Human respiratory syncytial virus (RSV) is the leading viral cause of severe lower respiratory disease in young children worldwide. As part of a genome-wide siRNA screen, we recently discovered that actin-related protein 2 (ARP2) is a host factor in the RSV replication cycle. ARP2 is a major constituent of the ARP2/3 complex, which catalyzes actin polymerization involved in cell morphology and motility. In the course of investigating this finding, we also found that RSV infection of human lung epithelial A459 cells induced filopodia formation and stimulated cell motility. The increase in filopodia formation was due, at least in part, to the expression of the RSV fusion F protein. Filopodia formation and increased cell motility appeared to shuttle RSV particles to nearby uninfected cells, facilitating virus cell-to-cell spread. ARP2 depletion did not reduce RSV entry or gene expression early in infection, but reduced subsequent virus production, filopodia formation, cell motility, and viral spread. Thus, the RSV F protein, ARP2-mediated actin nucleation, filopodia formation, and cell mobility all contribute to previously unrecognized mechanisms for RSV cell-to-cell spread that may promote RSV pathogenesis.","publicationTypes":["LettersAndComments","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The RSV F protein, ARP2-mediated actin nucleation, filopodia formation, and cell mobility all contribute to previously unrecognized mechanisms for RSV cell-to-cell spread that may promote RSV pathogenesis."}},"tag":"EXP"},{"id":236,"details":{"paperId":"465b8b5834703c012339e723ed98ba77cd7f8a73","externalIds":{"MAG":"1990464397","DOI":"10.1099/vir.0.032987-0","CorpusId":"12234950","PubMed":"21632562"},"title":"A novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus NS1 protein.","abstract":"Human respiratory syncytial virus (RSV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN)-dependent signalling, as well as IFN synthesis. RSV non-structural protein NS1 plays a significant role in this inhibition; however, the mechanism(s) responsible is not fully known. The transcription factor interferon regulatory factor (IRF)-3 is essential for viral-induced IFN-β synthesis. In this study, we found that NS1 protein inhibits IRF-3-dependent gene transcription in constitutively active IRF-3 overexpressing cells, demonstrating that NS1 directly targets IRF-3. Our data also demonstrate that NS1 associates with IRF-3 and its transcriptional coactivator CBP, leading to disrupted association of IRF-3 to CBP and subsequent reduced binding of IRF-3 to the IFN-β promoter without blocking viral-induced IRF-3 phosphorylation, nuclear translocation and dimerization, thereby identifying a novel molecular mechanism by which RSV inhibits IFN-β synthesis.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV non-structural protein NS1 protein inhibits IRF-3-dependent gene transcription in constitutively active IRf-3 overexpressing cells, demonstrating that NS1 directly targets IRF, which is essential for viral-induced IFN-β synthesis."}},"tag":"EXP"},{"id":224,"details":{"paperId":"5d8a27676702dfdc66de4cf20d3b9fa346e71078","externalIds":{"MAG":"2010004830","DOI":"10.1099/0022-1317-77-4-649","CorpusId":"37977108","PubMed":"8627253"},"title":"A point mutation in the F1 subunit of human respiratory syncytial virus fusion glycoprotein blocks its cell surface transport at an early stage of the exocytic pathway.","abstract":"Vaccinia virus recombinants expressing either wild-type or mutant forms of human respiratory syncytial (RS) virus (Long strain) fusion (F) glycoprotein were obtained. Proteolytic processing of the precursor, F0, and cell surface transport of the F glycoprotein were unaffected in the recombinants, except in those that contained the replacement Phe --> Ser at position 237 of the F1 subunit. In recombinants containing this mutation, either alone or in combination with others, the traffic of the F molecule was arrested at some intermediate step of its transport to the cell surface and, consequently, the endoproteolytic cleavage of the F0 precursor was inhibited. Immunofluorescence staining of infected cells and endoglycosidase H (Endo-H) sensitivity assays indicated that the arrest occurred before the mid-Golgi compartment. Dimerization and folding of the F protein were also affected by the Phe237 --> Ser substitution. Other amino acid replacements at positions 236 or 237 of the F1 subunit had various effects upon F0 maturation. These results are discussed in terms of the maturation requirements for the RS virus F molecule.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Recombinia virus recombinants expressing either wild-type or mutant forms of human respiratory syncytial (RS) virus (Long strain) fusion (F) glycoprotein were obtained and the maturation requirements for the RS virus F molecule were discussed."}},"tag":"EXP"},{"id":456,"details":{"paperId":"fe53e5ca071be4ba98a8366548192fc00d35333b","externalIds":{"MAG":"2936162970","PubMedCentral":"6504078","DOI":"10.1371/journal.ppat.1007707","CorpusId":"122379467","PubMed":"30995283"},"title":"A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes","abstract":"Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of a subset of RSV cbDVG species. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cBDVGs is not a stochastic process."}},"tag":"EXP"},{"id":189,"details":{"paperId":"40c0a5664f9e6fb79e6e8be2276d6b3e535c7831","externalIds":{"MAG":"2559226145","DOI":"10.1080/21505594.2016.1265725","CorpusId":"1487965","PubMed":"27911218"},"title":"Aberrant T cell immunity triggered by human Respiratory Syncytial Virus and human Metapneumovirus infection","abstract":"ABSTRACT Human Respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are the two major etiological viral agents of lower respiratory tract diseases, affecting mainly infants, young children and the elderly. Although the infection of both viruses trigger an antiviral immune response that mediate viral clearance and disease resolution in immunocompetent individuals, the promotion of long-term immunity appears to be deficient and reinfection are common throughout life. A possible explanation for this phenomenon is that hRSV and hMPV, can induce aberrant T cell responses, which leads to exacerbated lung inflammation and poor T and B cell memory immunity. The modulation of immune response exerted by both viruses include different strategies such as, impairment of immunological synapse mediated by viral proteins or soluble factors, and the induction of pro-inflammatory cytokines by epithelial cells, among others. All these viral strategies contribute to the alteration of the adaptive immunity in order to increase the susceptibility to reinfections. In this review, we discuss current research related to the mechanisms underlying the impairment of T and B cell immune responses induced by hRSV and hMPV infection. In addition, we described the role each virulence factor involved in immune modulation caused by these viruses.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Current research related to the mechanisms underlying the impairment of T and B cell immune responses induced by hRSV and hMPV infection are discussed and the role each virulence factor involved in immune modulation caused by these viruses is described."}},"tag":"EXP"},{"id":204,"details":{"paperId":"95fd3990adcd11e86a90afe7ac3a4b106c1f3d56","externalIds":{"PubMedCentral":"9615430","DOI":"10.1093/ve/veac091","CorpusId":"249133657","PubMed":"36325033"},"title":"Accumulation of copy-back viral genomes during respiratory syncytial virus infection is preceded by diversification of the copy-back viral genome population followed by selection","abstract":"RNA viruses generate non-standard viral genomes during their replication, including viral genomes of the copy-back (cbVG) type that cannot replicate in the absence of a standard virus. cbVGs play a crucial role in shaping virus infection outcomes due to their ability to interfere with virus replication and induce strong immune responses. However, despite their critical role during infection, the principles that drive the selection and evolution of cbVGs within a virus population are poorly understood. As cbVGs are dependent on the virus replication machinery to be generated and replicated, we hypothesized that host factors that affect virus replication exert selective pressure on cbVGs and drive their evolution within a virus population. To test this hypothesis, we used respiratory syncytial virus (RSV) as model and took an experimental evolution approach by serially passaging RSV in immune competent A549 control and immune deficient A549 STAT1 KO cells which allow higher levels of virus replication. As predicted, we observed that virus populations accumulated higher amounts of cbVGs in the more permissive A549 STAT1 KO cells over time but, unexpectedly, the predominant cbVG species after passages in the two conditions were different. While A549 STAT1 KO cells accumulated relatively short cbVGs, A549 control cells mainly contained cbVGs of much longer predicted size that have not been described previously. These long cbVGs were the first species generated in both cell lines in vitro and the predominant ones observed in samples from RSV infected patients. Although high replication levels are associated with cbVG generation and accumulation, our data show that high levels of virus replication are critical for cbVG population diversification, a process that preceded the generation of shorter cbVGs that selectively accumulated over time. Taken together, we show that selection and evolution of cbVGs within a virus population is shaped by how resistant (low replication) or permissive (high replication) a host is to RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that selection and evolution of cbVGs within a virus population is shaped by how resistant or permissive a host is to RSV, and that high levels of virus replication are critical for cbVG population diversification."}},"tag":"EXP"},{"id":450,"details":{"paperId":"d384dfba2d980713d8398416165b22705215d6e5","externalIds":{"MAG":"2559878375","PubMedCentral":"5142808","DOI":"10.1371/journal.ppat.1006062","CorpusId":"16086968","PubMed":"27926942"},"title":"Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread","abstract":"Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread are identified and a new role for the RSV F protein, driving filopoda induction and virus spread is identified."}},"tag":"EXP"},{"id":296,"details":{"paperId":"9e3458a3f32eea3067765b07a583967089b3e613","externalIds":{"PubMedCentral":"9555202","DOI":"10.1128/jvi.01297-22","CorpusId":"252216271","PubMed":"36102648"},"title":"An Unexpected Encounter: Respiratory Syncytial Virus Nonstructural Protein 1 Interacts with Mediator Subunit MED25","abstract":"Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. ABSTRACT Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.","publicationTypes":["JournalArticle","Review"],"tldr":{"model":"tldr@v2.0.0","text":"The interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection, and a role in innate anti-RSV defense for this Mediator complex subunit is proposed."}},"tag":"EXP"},{"id":8,"details":{"paperId":"ffc76c110b4e356b3be76bf636194e14abb8ee46","externalIds":{"MAG":"2016867278","DOI":"10.1006/VIRO.1994.1245","CorpusId":"5992795","PubMed":"8178462"},"title":"Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion.","abstract":"Recombinant expression of the human respiratory syncytial virus (RSV) fusion (F) glycoprotein, receptor-binding glycoprotein (G), and small hydrophobic (SH) protein was performed to determine the role(s) of these proteins in syncytia formation. These studies used a vaccinia virus expressing the bacteriophage (T7) RNA polymerase gene and plasmid vectors containing the RSV genes under the control of a T7 promoter. Within the context of this expression system, expression of any individual RSV gene, or coexpression of F+G genes, did not elicit the formation of syncytia. However, at plasmid input levels which were 10-fold higher than those normally used, coexpression of F+G induced low but detectable levels of cell fusion. In contrast, coexpression of F, G, and SH together elicited extensive cell fusion resembling that of an authentically infected cell monolayer. In addition, coexpression of F and SH elicited significant cell fusion, although to a lesser extent than was observed when G was included. Cell fusion induced by coexpression of F+SH was found to be specific to the RSV proteins, since coexpression of SH with the analogous F proteins from human parainfluenza virus type 3, human parainfluenza virus type 2, Sendai virus, or simian virus type 5 (SV5) did not elicit cell fusion. Finally, coexpression of the SV5 SH protein with the RSV or SV5 glycoproteins also failed to induce syncytia, suggesting type-specific restrictions between the two sets of viral proteins.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Cell fusion induced by coexpression of F+SH was found to be specific to the RSV proteins, since co expression of SH with the analogous F proteins from human parainfluenza virus type 2, Sendai virus, or simian virus type 5 (SV5) did not elicit cell fusion."}},"tag":"EXP"},{"id":348,"details":{"paperId":"dd8d4a0cd85009e516a371b6e0f6431dbb3feec9","externalIds":{"MAG":"2147880851","DOI":"10.1128/jvi.71.7.4944-4953.1997","CorpusId":"245032","PubMed":"9188557"},"title":"Analysis of the gene start and gene end signals of human respiratory syncytial virus: quasi-templated initiation at position 1 of the encoded mRNA","abstract":"The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain. The GS signal, which is the start site for mRNA synthesis, is highly conserved for the first nine genes: 3'-CCCCGUUUA(U/C) (negative sense). This conserved version of the signal was analyzed by \"saturation\" mutagenesis, in which all 10 positions, as well as one downstream and one upstream position, were changed one at a time into each of the other three nucleotides. Most of the positions appear to contribute to the signal: positions 1, 3, 6, 7, and, in particular, 9 were the most sensitive, whereas position 5 was relatively insensitive. The effect of nucleotide substitution in the first position of the signal was examined further by cDNA cloning and sequence analysis of the residual mRNA which was produced. For the two mutants examined (1C to U, and 1C to A), the site of initiation was unchanged. However, the mRNAs were dimorphic with regard to the assignment of the 5'-terminal nucleotide: two-thirds contained the predicted mutant substitution, and one-third contained the parental assignment. Intracellular minigenome contained only the mutant assignment, indicating that the heterogeneity was at the level of transcription by the RSV polymerase. This suggests that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment, a phenomenon which we dubbed quasi-templated initiation. The GS signal of the L gene, encoding the 10th RSV mRNA, contains three differences (3'-CCCUGUUUUA) compared to the conserved version. It was shown to be equal in efficiency to the conserved version. This was unexpected, since the saturation mutagenesis described above indicated that U in place of A at position 9 should be highly inhibitory. Instead, the A at position 10 of the L GS signal was found to be critical for activity, indicating that an essential A residue indeed was present in both versions of the GS signal but that its spacing differed. The GE signal, which directs termination and polyadenylation, has more sequence diversity in nature than does the GS signal. The naturally occurring GE signals of strain A2 were compared by their individual incorporation into a dicistronic minigenome. They were similar in the ability to produce translatable mRNA except in the cases of NS1 and NS2, which were approximately 60% as efficient.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The gene start (GS) and gene end (GE) transcription signals of human respiratory syncytial virus (RSV) strain A2 were analyzed in helper-dependent monocistronic and dicistronic minireplicons which were complemented by a standard RSV strain, suggesting that the templated mutant assignment at position 1 can sometimes be overridden by an innate preference for the parental assignment."}},"tag":"EXP"},{"id":162,"details":{"paperId":"75f8b52741907d82edb168d3a2596e184b07790e","externalIds":{"MAG":"1994806657","DOI":"10.1073/pnas.2434327100","CorpusId":"33108029","PubMed":"14630951"},"title":"Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells","abstract":"Respiratory syncytial virus (RSV) is the major viral cause of serious lower respiratory tract illness in infants and young children worldwide. RSV infection is limited to the superficial layers of the respiratory epithelium in immunocompetent individuals. Consistent with this in vivo observation, we and others have found that RSV buds preferentially from the apical surface of infected polarized epithelial cells. In contrast, directional budding is not observed in nonpolarized human epithelial cells. These findings suggest that RSV uses specific cellular trafficking pathways to accomplish viral replication. The host cell proteins that regulate directional budding of RSV are undefined. Apical sorting of cellular proteins in polarized epithelial cells involves the apical recycling endosome (ARE). To investigate whether ARE-mediated protein sorting plays a role during RSV replication, we expressed a fragment of the myosin Vb tail that functions as a dominant negative inhibitor of ARE-mediated protein sorting in polarized Madin-Darby canine kidney cells. When these cells were infected with RSV, a >9,000-fold reduction in viral yield was observed. A similar effect on virus replication was observed when a carboxyl-terminal fragment of another ARE-associated protein, the Rab11 family interacting protein 1, was expressed in Madin-Darby canine kidney cells. These data suggest that RSV requires proper ARE-mediated protein sorting for efficient egress from the apical surface of polarized epithelial cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that RSV requires proper ARE-mediated protein sorting for efficient egress from the apical surface of polarized epithelial cells, which suggests thatRSV uses specific cellular trafficking pathways to accomplish viral replication."}},"tag":"EXP"},{"id":210,"details":{"paperId":"b21e22c39853e09f135c5147280ef7a2361405c1","externalIds":{"MAG":"2140223829","DOI":"10.1099/0022-1317-68-6-1705","CorpusId":"15419792","PubMed":"3585282"},"title":"Appearance of a soluble form of the G protein of respiratory syncytial virus in fluids of infected cells.","abstract":"We examined the antigenic reactivities and virion associations of glycoproteins that were released into the culture fluids of cells infected with respiratory syncytial (RS) virus. Culture fluids and cell extracts were obtained from cells 24 to 30 h after they were infected with the Long strain of RS virus. Radioimmune precipitation of [3H]glucosamine-labelled glycoproteins by large glycoprotein (G)-specific or fusion protein (F)-specific monoclonal antibodies (MAbs) revealed that the G, F1 and F2 proteins were present in cell extracts but only the G protein was clearly evident in culture fluids. A glycoprotein (Mr 43K) which may be a precursor or a breakdown product of the G protein was also precipitated by the G-specific MAb from cell extracts and culture fluids. The G protein in culture fluids was slightly smaller (Mr 82K) than the G protein in cell extracts (Mr 88K). An abundant or heavily labelled, 18K glycoprotein in the fluids of virus-infected but not of mock-infected cells was weakly precipitated by the F-specific MAb; this suggested that the 18K protein shares epitopes with the fusion protein of RS virus. The absence of F1 and F2 polypeptides from culture fluids is evidence that the cells, which contained an abundance of these proteins, were intact. To determine whether any of the viral glycoproteins released by infected cells were soluble (non-virion-associated), culture fluid was subjected to rate zonal centrifugation in a 10 to 50% sucrose gradient. An assay of fractions using a MAb-capture ELISA for the nucleocapsid (N) and F proteins revealed a peak of activity, due to virions, in the centre of the gradient, and a strong signal for the N protein at the top of the gradient suggesting that N protein was released from intact cells. Radioimmune precipitation of glycoproteins from the fractions at the top of the gradient using a hyperimmune guinea-pig serum revealed the G protein and a heterogeneous band which had the electrophoretic mobility of the 43K protein. Neither the F1 nor the F2 protein was present in these fractions thus suggesting that virions had remained intact. These results showed that a soluble form of the G protein of RS virus is released into the culture fluids of intact, infected cells. Several theories concerning viral and non-viral origins for the 18K protein are discussed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results showed that a soluble form of the G protein of RS virus is released into the culture fluids of intact, infected cells."}},"tag":"EXP"},{"id":278,"details":{"paperId":"6ad3fd26672063839189164d76b0778ad5b732ae","externalIds":{"MAG":"2128028490","DOI":"10.1128/JVI.00343-08","CorpusId":"7722871","PubMed":"18579594"},"title":"Association of Respiratory Syncytial Virus M Protein with Viral Nucleocapsids Is Mediated by the M2-1 Protein","abstract":"ABSTRACT Cytoplasmic inclusions in respiratory syncytial virus-infected cells comprising viral nucleocapsid proteins (L, N, P, and M2-1) and the viral genome are sites of viral transcription. Although not believed to be necessary for transcription, the matrix (M) protein is also present in these inclusions, and we have previously shown that M inhibits viral transcription. In this study, we have investigated the mechanisms for the association of the M protein with cytoplasmic inclusions. Our data demonstrate for the first time that the M protein associates with cytoplasmic inclusions via an interaction with the M2-1 protein. The M protein colocalizes with M2-1 in the cytoplasm of cells expressing only the M and M2-1 proteins and directly interacts with M2-1 in a cell-free binding assay. Using a cotransfection system, we confirmed that the N and P proteins are sufficient to form cytoplasmic inclusions and that M2-1 localizes to these inclusions; additionally, we show that M associates with cytoplasmic inclusions only in the presence of the M2-1 protein. Using truncated mutants, we show that the N-terminal 110 amino acids of M mediate the interaction with M2-1 and the subsequent association with nucleocapsids. The interaction of M2-1 with M and, in particular, the N-terminal region of M may represent a target for novel antivirals that block the association of M with nucleocapsids, thereby inhibiting virus assembly.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data demonstrate for the first time that the M protein associates with cytoplasmic inclusions via an interaction with the M2-1 protein, which may represent a target for novel antivirals that block the association of M with nucleocapsids, thereby inhibiting virus assembly."}},"tag":"EXP"},{"id":34,"details":{"paperId":"d84007911895b743a49dfca1ee0ada117f09745a","externalIds":{"MAG":"2008848537","DOI":"10.1007/s00705-003-0183-9","CorpusId":"20941856","PubMed":"14689285"},"title":"Association of matrix protein of respiratory syncytial virus with the host cell membrane of infected cells","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The possible association of respiratory syncytial virus (RSV) matrix (M) protein with the plasma membrane of infected cells is investigated and it is found that RSV uses lipid rafts for assembly and budding."}},"tag":"EXP"},{"id":9,"details":{"paperId":"d67cd02ca5e2ac953a7ca301f2e884056a8f7236","externalIds":{"MAG":"2031490666","DOI":"10.1006/VIRO.1994.1623","CorpusId":"45805000","PubMed":"7975241"},"title":"Bacterial expression of human respiratory syncytial viral phosphoprotein P and identification of Ser237 as the site of phosphorylation by cellular casein kinase II.","abstract":"The phosphoprotein P gene of human respiratory syncytial virus has been cloned and the protein expressed in Escherichia coli. The expressed protein was soluble, unphosphorylated, and constituted approximately 10% of the total bacterial protein. Electrophoretic and antigenic analyses demonstrated the identity of the recombinant protein with viral P protein and P protein synthesized in reticulocyte lysates. Purified recombinant P protein was efficiently phosphorylated in vitro by purified native as well as recombinant casein kinase II (CKII) or by the CKII activity in uninfected cell extracts. Through deletions and site-directed mutagenesis, the site of CKII phosphorylation was mapped to a single serine residue (Ser237) near the C-terminal end of the P protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Electrophoretic and antigenic analyses demonstrated the identity of the recombinant protein with viral P protein and P protein synthesized in reticulocyte lysates and the site of CKII phosphorylation was mapped to a single serine residue near the C-terminal end of the P protein."}},"tag":"EXP"},{"id":150,"details":{"paperId":"6858f23c76d4c9cc56a1a01251cb3e380f3b75ad","externalIds":{"MAG":"2043192321","DOI":"10.1046/j.1462-5822.2003.00313.x","CorpusId":"28548829","PubMed":"12969373"},"title":"Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response","abstract":"Respiratory syncytial virus (RSV) is the most common cause of severe lower respiratory tract infection in infants and the elderly. There is currently no effective antiviral treatment for the infection, but advances in our understanding of RSV uptake, especially the role of surfactant proteins, the attachment protein G and the fusion protein F, as well as the post‐binding events, have revealed potential targets for new therapies and vaccine development. RSV infection triggers an intense inflammatory response, mediated initially by the infected airway epithelial cells and antigen‐presenting cells. Humoral and cell‐mediated immune responses are important in controlling the extent of infection and promoting viral clearance. The initial innate immune response may play a critical role by influencing the subsequent adaptive response generated. This review summarizes our current understanding of RSV binding and uptake in mammalian cells and how these initial interactions influence the subsequent innate immune response generated.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Current understanding of RSV binding and uptake in mammalian cells and how these initial interactions influence the subsequent innate immune response generated are summarized."}},"tag":"EXP"},{"id":122,"details":{"paperId":"14315524d00b73bdc9f69f10b0e9262a0098e795","externalIds":{"MAG":"3083376108","DOI":"10.1021/acsinfecdis.0c00554","CorpusId":"221502438","PubMed":"32886480"},"title":"Biochemical characterization of respiratory syncytial virus RNA-dependent RNA polymerase complex.","abstract":"RNA-dependent RNA polymerases (RdRP) from non-segmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP. We determined that the intact L protein is essential for RdRP activity and in truncated L protein constructs RdRP activity is abrogated due to their deficiency in RNA template binding. These results are in agreement with the observation of an RNA template-binding tunnel at the interface of RdRP and capping domains in RSV and vesicular stomatitis virus (VSV) L protein cryo-EM structures. We also describe non-radiometric assays for measuring RNA binding and RNA polymerization activity of RSV RdRP which are amenable to compound screening and profiling.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is determined that the intact L protein is essential for RdRP activity and in truncated L protein constructs Rd RP activity is abrogated due to their deficiency in RNA template binding."}},"tag":"EXP"},{"id":180,"details":{"paperId":"e0ce1b57618e88ed5e441eb2d5ee502fd40b36be","externalIds":{"MAG":"2908682429","DOI":"10.1074/jbc.RA118.006453","CorpusId":"58600107","PubMed":"30626736"},"title":"Biochemical characterization of the respiratory syncytial virus N0-P complex in solution","abstract":"As all the viruses belonging to the Mononegavirales order, the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N0-P complex. We have previously demonstrated that residues 1–30 of P specifically bind to N0. Here, to isolate a stable N0-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N0-P–like complex, a deletion of the first 30 N-terminal residues of N (NΔ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N0-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N0-P complexes. In both complexes, the P40-binding sites on N0 appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N0-P for the development of RSV antivirals.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Structural models of the RSV N0-P are generated with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants, which suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV), and the P40-binding sites on N0 appear to be similar."}},"tag":"EXP"},{"id":240,"details":{"paperId":"aa678016e3ff2193ac4c6d3e3b25baa9142de034","externalIds":{"MAG":"2105881372","DOI":"10.1099/VIR.0.79830-0","CorpusId":"6042785","PubMed":"15166449"},"title":"Biochemical characterization of the respiratory syncytial virus P-P and P-N protein complexes and localization of the P protein oligomerization domain.","abstract":"The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase-transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P-P and N-P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120-150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N."}},"tag":"EXP"},{"id":303,"details":{"paperId":"5d95fe890b82761811abca19fbc7b1aea83900a6","externalIds":{"MAG":"3087584985","DOI":"10.1128/JVI.01505-20","CorpusId":"221769700","PubMed":"32938771"},"title":"Biophysical and Dynamic Characterization of Fine-Tuned Binding of the Human Respiratory Syncytial Virus M2-1 Core Domain to Long RNAs","abstract":"The main outcome is the molecular description of the fine-tuned binding of the cdM2-1/RNA complex and the provision of evidence that the domain alone has unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Human respiratory syncytial virus (hRSV), an orthopneumovirus, stands out for the unique role of its M2-1 protein as a transcriptional antitermination factor able to increase RNA polymerase processivity. ABSTRACT The human respiratory syncytial virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here, the first evidence that the hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs is presented and the biophysical and dynamic characterization of the cdM2-1/RNA complex is provided. The main contact region of cdM2-1 with RNA was the α1-α2-α5-α6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their termini over the domain. The α2-α3 and α3-α4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding, even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to the unraveling interaction aspects necessary for M2-1 activity. IMPORTANCE The main outcome is the molecular description of the fine-tuned binding of the cdM2-1/RNA complex and the provision of evidence that the domain alone has unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Human respiratory syncytial virus (hRSV), an orthopneumovirus, stands out for the unique role of its M2-1 protein as a transcriptional antitermination factor able to increase RNA polymerase processivity.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to the unraveling interaction aspects necessary for M2-0 activity and evidence that the domain alone has unfolding activity for long RNAs."}},"tag":"EXP"},{"id":251,"details":{"paperId":"f5f2099e4b54c08788f30e790542ce52da5b8fad","externalIds":{"MAG":"3044165734","DOI":"10.1101/2020.07.22.216952","CorpusId":"220835773"},"title":"Biophysical and dynamic characterization of a fine-tuned binding of the human Respiratory Syncytial Virus M2-1 core domain to long RNAs","abstract":"The human Respiratory Syncytial Virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here it is presented the first evidence that hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs, as well as a biophysical and dynamic characterization of the cdM2-1/RNA complex. The main contact region of cdM2-1 with RNA was the α1–α2–α5–α6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their terminals over the domain. The α2–α3 and α3–α4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to unraveling interaction aspects necessary to M2-1 activity. IMPORTANCE The main outcome is the molecular description of a fine-tuned binding of the cdM2-1/RNA complex and the evidence that the domain alone has an unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Orthopneumovirus, as the human Respiratory Syncytial Virus (hRSV), stands out for the unique role of M2-1 as a transcriptional antitermination factor able to increase the RNA polymerase processivity.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The main outcome is the molecular description of a fine-tuned binding of the cdM2-1/RNA complex and the evidence that the domain alone has an unfolding activity for long RNAs, essential in the understanding of the function in the full-length protein."}},"tag":"EXP"},{"id":108,"details":{"paperId":"86e004e7b3cf4291d93cea85f312dab2215728ba","externalIds":{"MAG":"2058832585","DOI":"10.1016/S0006-291X(03)00197-9","CorpusId":"20017784","PubMed":"12615056"},"title":"Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that both HR1 and HR2 of hRSV F protein give strong inhibition on virus fusion in some members of paramyxovirus and they form stable six-helix bundle in vitro with both in the fusion protein form."}},"tag":"EXP"},{"id":223,"details":{"paperId":"385887b391981070a50f3cf3332bcc85ee810197","externalIds":{"MAG":"2131089277","DOI":"10.1099/0022-1317-76-2-425","CorpusId":"13631787","PubMed":"7844563"},"title":"C-terminal phosphorylation of human respiratory syncytial virus P protein occurs mainly at serine residue 232.","abstract":"To determine which human respiratory syncytial virus (HRSV) P protein serine residues are modified by cellular protein kinase(s), several mutated versions of P protein were expressed in the absence of other viral proteins. Mutations at serines 232 or 232 and 237 drastically reduced the extent of phosphorylation P protein in vivo. Serine 232 is the main site of modification and is also essential for in vitro phosphorylation by casein kinase II. Additional in vivo phosphorylation was also detected in the region containing serines 116, 117 and 119.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"To determine which human respiratory syncytial virus P protein serine residues are modified by cellular protein kinase(s), several mutated versions of P protein were expressed in the absence of other viral proteins."}},"tag":"EXP"},{"id":153,"details":{"paperId":"ce1891ca22bd03949f08a67f2ac3a3022fce71d4","externalIds":{"MAG":"2158922816","DOI":"10.1073/PNAS.0403812101","CorpusId":"35160827","PubMed":"15496474"},"title":"CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus.","abstract":"Angiotensin-converting enzyme 2 (ACE2) is a receptor for SARS-CoV, the novel coronavirus that causes severe acute respiratory syndrome [Li, W. Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., et al. (2003) Nature 426, 450-454]. We have identified a different human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV. A human lung cDNA library in vesicular stomatitis virus G pseudotyped retrovirus was transduced into Chinese hamster ovary cells, and the cells were sorted for binding of soluble SARS-CoV spike (S) glycoproteins, S(590) and S(1180). Clones of transduced cells that bound SARS-CoV S glycoprotein were inoculated with SARS-CoV, and increases in subgenomic viral RNA from 1-16 h or more were detected by multiplex RT-PCR in four cloned cell lines. Sequencing of the human lung cDNA inserts showed that each of the cloned cell lines contained cDNA that encoded human CD209L, a C-type lectin (also called L-SIGN). When the cDNA encoding CD209L from clone 2.27 was cloned and transfected into Chinese hamster ovary cells, the cells expressed human CD209L glycoprotein and became susceptible to infection with SARS-CoV. Immunohistochemistry showed that CD209L is expressed in human lung in type II alveolar cells and endothelial cells, both potential targets for SARS-CoV. Several other enveloped viruses including Ebola and Sindbis also use CD209L as a portal of entry, and HIV and hepatitis C virus can bind to CD209L on cell membranes but do not use it to mediate virus entry. Our data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in virus infection and pathogenesis.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that the large S glycoprotein of SARS-CoV may use both ACE2 and CD209L in virus infection and pathogenesis, and several other enveloped viruses including Ebola and Sindbis also useCD209L as a portal of entry."}},"tag":"EXP"},{"id":132,"details":{"paperId":"2a84947095a18cbefb4c5179f9d1529c14d28b1c","externalIds":{"MAG":"1587614700","DOI":"10.1038/90675","CorpusId":"11545720","PubMed":"11477410"},"title":"CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"CX3C chemokine mimicry for the G glycoprotein of respiratory syncytial virus (RSV) is reported and binding to CX3CR1—the specific receptor for the C X3CChemokine fractalkine—and induction of leukocyte chemotaxis are shown and it is shown that CX 3CR1 facilitates RSV infection of cells."}},"tag":"EXP"},{"id":270,"details":{"paperId":"40b6357833fb373e694207a5616007bb24ce22be","externalIds":{"DOI":"10.1128/JVI.00095-21","CorpusId":"232303332","PubMed":"33731455"},"title":"CX3CR1 Engagement by Respiratory Syncytial Virus Leads to Induction of Nucleolin and Dysregulation of Cilium-Related Genes","abstract":"Respiratory syncytial virus (RSV) has an enormous impact on infants and the elderly, including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. ABSTRACT Respiratory syncytial virus (RSV) contains a conserved CX3C motif on the ectodomain of the G protein. The motif has been indicated as facilitating attachment of the virus to the host, initiating infection via the human CX3CR1 receptor. The natural CX3CR1 ligand, CX3CL1, has been shown to induce signaling pathways resulting in transcriptional changes in the host cells. We hypothesize that binding of RSV to CX3CR1 via CX3C leads to transcriptional changes in host epithelial cells. Using transcriptomic analysis, the effect of CX3CR1 engagement by RSV was investigated. Normal human bronchial epithelial (NHBE) cells were infected with RSV virus containing either wild-type (WT) G protein or a mutant virus containing a CX4C mutation in the G protein. RNA sequencing was performed on mock-infected and 4-days-postinfected cultures. NHBE cultures were also treated with purified recombinant wild-type A2 G protein. Here, we report that RSV infection resulted in significant changes in the levels of 766 transcripts. Many nuclear-associated proteins were upregulated in the WT group, including nucleolin. In contrast, cilium-associated genes, including CC2D2A and CFAP221 (PCDP1), were downregulated. The addition of recombinant G protein to the culture led to the suppression of cilium-related genes while also inducing nucleolin. Mutation of the CX3C motif (CX4C) reversed these effects on transcription, decreasing nucleolin induction and lessening the suppression of cilium-related transcripts in culture. Furthermore, immunohistochemical staining demonstrated decreases in ciliated cells and altered morphology. Therefore, it appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with the function of cilia. IMPORTANCE Respiratory syncytial virus (RSV) has an enormous impact on infants and the elderly, including increased fatality rates and potential for causing lifelong lung problems. Humans become infected with RSV through the inhalation of viral particles exhaled from an infected individual. These virus particles contain specific proteins that the virus uses to attach to human ciliated lung epithelial cells, initiating infection. Two viral proteins, G protein and F protein, have been shown to bind to human CX3CR1and nucleolin, respectively. Here, we show that the G protein induces nucleolin and suppresses gene transcripts specific to ciliated cells. Furthermore, we show that mutation of the CX3C-motif on the G protein, CX4C, reverses these transcriptional changes.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It appears that engagement of CX3CR1 leads to induction of genes necessary for RSV entry as well as dysregulation of genes associated with the function of cilia."}},"tag":"EXP"},{"id":441,"details":{"paperId":"aec1f2ec6484353663caf65f08a85d8f93491f67","externalIds":{"PubMedCentral":"4479564","MAG":"1527926071","DOI":"10.1371/journal.pone.0130517","CorpusId":"15184932","PubMed":"26107373"},"title":"CX3CR1 Is Expressed in Differentiated Human Ciliated Airway Cells and Co-Localizes with Respiratory Syncytial Virus on Cilia in a G Protein-Dependent Manner","abstract":"Respiratory syncytial virus (RSV) is the principal cause of bronchiolitis in infants and a significant healthcare problem. The RSV Glycoprotein (G) mediates attachment of the virus to the cell membrane, which facilitates interaction of the RSV Fusion (F) protein with nucleolin, thereby triggering fusion of the viral and cellular membranes. However, a host protein ligand for G has not yet been identified. Here we show that CX3CR1 is expressed in the motile cilia of differentiated human airway epithelial (HAE) cells, and that CX3CR1 co-localizes with RSV particles. Upon infection, the distribution of CX3CR1 in these cells is significantly altered. Complete or partial deletion of RSV G results in viruses binding at least 72-fold less efficiently to cells, and reduces virus replication. Moreover, an antibody targeting an epitope near the G protein’s CX3CR1-binding motif significantly inhibits binding of the virus to airway cells. Given previously published evidence of the interaction of G with CX3CR1 in human lymphocytes, these findings suggest a role for G in the interaction of RSV with ciliated lung cells. This interpretation is consistent with past studies showing a protective benefit in immunizing against G in animal models of RSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy. CX3CR1 expression in lung epithelial cells may also have implications for other respiratory diseases such as asthma.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Findings suggest a role for G in the interaction of RSV with ciliated lung cells, consistent with past studies showing a protective benefit in immunizing against G in animal models ofRSV infection, and would support targeting the CX3CR1-G protein interaction for prophylaxis or therapy."}},"tag":"EXP"},{"id":268,"details":{"paperId":"eac3fe5d509e2920c9da93851e9c94899c963a4f","externalIds":{"DOI":"10.1128/JVI.00010-21","CorpusId":"235202217","PubMed":"34037420"},"title":"CX3CR1 Is a Receptor for Human Respiratory Syncytial Virus in Cotton Rats","abstract":"The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. ABSTRACT Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo, we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results indicate that CX3CR1 functions as a receptor for RSV in cotton rats, and in combination with data from human airway epithelial cell cultures, strongly suggest that Cx3 CR1 is a primary receptor for naturally acquired RSV infection."}},"tag":"EXP"},{"id":138,"details":{"paperId":"7712ca5b67c625b00912615a3a92e54db9530e9a","externalIds":{"MAG":"2988100176","DOI":"10.1038/s41390-019-0677-0","CorpusId":"208039423","PubMed":"31726465"},"title":"CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"CX3CR1 is present in the airways of pediatric subjects where it may serve as a receptor for RSV infection and appears to play a mechanistic role in mediating viral infection of pediatric airway epithelial cells in vitro."}},"tag":"EXP"},{"id":235,"details":{"paperId":"ee29a03de28368db16e04ac4b2de35f1b19beae4","externalIds":{"MAG":"2114446982","DOI":"10.1099/vir.0.000218","CorpusId":"25517019","PubMed":"26297201"},"title":"CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells.","abstract":"Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES, IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1."}},"tag":"EXP"},{"id":267,"details":{"paperId":"95619821c186d146a7aaf4631c060f6a5fe17fed","externalIds":{"MAG":"1501551172","DOI":"10.1128/jcm.26.1.139-141.1988","CorpusId":"7458980","PubMed":"3343306"},"title":"Calcium requirement for syncytium formation in HEp-2 cells by respiratory syncytial virus","abstract":"Respiratory syncytial virus (RSV) grown in HEp-2 cells in the absence of calcium did not induce cell fusion and syncytium formation. Although the infected cells contained viral antigens, the cytopathic effect (giant cell formation) typical for RSV was not observed in calcium-free cultures. Infectious virus yield was also slightly reduced (less than a one log10 reduction) in the absence of calcium. An analysis of viral proteins synthesized in both the presence and the absence of calcium revealed that the amount of fusion protein (F1) in calcium-free infected cultures was approximately one-third that in calcium-containing infected cultures. These results underscore the necessity of using calcium-containing growth medium for cell culture isolation and diagnosis of RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV grown in HEp-2 cells in the absence of calcium did not induce cell fusion and syncytium formation, highlighting the necessity of using calcium-containing growth medium for cell culture isolation and diagnosis of RSV."}},"tag":"EXP"},{"id":354,"details":{"paperId":"b3e1b1e6a4a8ce0f4983d894eb06f9183592e89a","externalIds":{"MAG":"2131522839","DOI":"10.1128/JVI.73.10.8384-8392.1999","CorpusId":"19520569","PubMed":"10482589"},"title":"Casein Kinase 2-Mediated Phosphorylation of Respiratory Syncytial Virus Phosphoprotein P Is Essential for the Transcription Elongation Activity of the Viral Polymerase; Phosphorylation by Casein Kinase 1 Occurs Mainly at Ser215 and Is without Effect","abstract":"ABSTRACT The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser232 in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts. A recombinant P protein in which Ser232 was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser215. In vitro, P protein could be phosphorylated by purified CK1 at Ser215 but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser215, Ser232, and Ser237 were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase is suggested."}},"tag":"EXP"},{"id":431,"details":{"paperId":"33284105ff291cdae6c2b0c78169c0f5890dd6f0","externalIds":{"MAG":"2583992088","PubMedCentral":"5358342","DOI":"10.1242/jcs.198853","CorpusId":"51801","PubMed":"28154158"},"title":"Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is an enveloped virus that assembles into filamentous virus particles on the surface of infected cells. Morphogenesis of RSV is dependent upon cholesterol-rich (lipid raft) membrane microdomains, but the specific role of individual raft molecules in RSV assembly is not well defined. Here, we show that RSV morphogenesis occurs within caveolar membranes and that both caveolin-1 and cavin-1 (also known as PTRF), the two major structural and functional components of caveolae, are actively recruited to and incorporated into the RSV envelope. The recruitment of caveolae occurred just prior to the initiation of RSV filament assembly, and was dependent upon an intact actin network as well as a direct physical interaction between caveolin-1 and the viral G protein. Moreover, cavin-1 protein levels were significantly increased in RSV-infected cells, leading to a virus-induced change in the stoichiometry and biophysical properties of the caveolar coat complex. Our data indicate that RSV exploits caveolae for its assembly, and we propose that the incorporation of caveolae into the virus contributes to defining the biological properties of the RSV envelope. Highlighted Article: The assembly of respiratory syncytial virus occurs in caveolar membranes and caveolar coat components are incorporated into the viral envelope.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV morphogenesis occurs within caveolar membranes and that both caveolin-1 and cavin-1 (also known as PTRF), the two major structural and functional components of caveolae, are actively recruited to and incorporated into the RSV envelope."}},"tag":"EXP"},{"id":263,"details":{"paperId":"9d03402697c6c1ccc92ea84d45da2d92db4071d8","externalIds":{"MAG":"2172117809","DOI":"10.1111/J.1574-6976.2006.00025.X","CorpusId":"38774208","PubMed":"16911040"},"title":"Central role of the respiratory syncytial virus matrix protein in infection.","abstract":"Respiratory syncytial virus is the major respiratory pathogen of infants and children worldwide, with no effective treatment or vaccine available. Steady progress has been made in understanding the respiratory syncytial virus life cycle and the consequences of infection, but many areas of respiratory syncytial virus biology remain poorly understood, including the role of subcellular localisation of respiratory syncytial virus gene products such as the matrix protein in the infected host cell. The matrix protein plays a central role in viral assembly and, intriguingly, has been observed to traffic into and out of the nucleus at specific times during the respiratory syncytial virus infectious cycle. Further, the matrix protein has been shown to be able to inhibit transcription, which may be a key to respiratory syncytial virus pathogenesis. This review will focus on the role of the matrix protein in respiratory syncytial virus infection and what is known of its nucleocytoplasmic trafficking, the understanding of which may lead to new therapeutic approaches to combat respiratory syncytial virus, and/or vaccine development.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The matrix protein plays a central role in viral assembly and, intriguingly, has been observed to traffic into and out of the nucleus at specific times during the respiratory syncytial virus infectious cycle."}},"tag":"EXP"},{"id":269,"details":{"paperId":"bae6a1639c94f1b20af71b15d7af19231dde24b4","externalIds":{"MAG":"2151572665","DOI":"10.1128/JVI.00058-12","CorpusId":"23589207","PubMed":"22623798"},"title":"Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein","abstract":"ABSTRACT The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTD and molecular modeling of the NNTD allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTD interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"GST pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain and define four potential candidate pockets on N as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTD interaction domain."}},"tag":"EXP"},{"id":116,"details":{"paperId":"209498c9e4dbe661917045f07bd7bfdace70074f","externalIds":{"MAG":"2035196516","DOI":"10.1016/S0168-1702(98)00042-2","CorpusId":"20597815","PubMed":"9725669"},"title":"Characterization of the interaction of the human respiratory syncytial virus phosphoprotein and nucleocapsid protein using the two-hybrid system.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data shows that the RSV P protein, while having no amino acid sequence identity with the equivalent P protein of other negative strand viruses, is likely to have similar structural and functional features."}},"tag":"EXP"},{"id":107,"details":{"paperId":"ba53a881a21f2ec0e48808c38d8d1453137ad811","externalIds":{"MAG":"2937661759","DOI":"10.1016/j.virusres.2019.04.006","CorpusId":"125218139","PubMed":"31004621"},"title":"Characterization of the role of N-glycosylation sites in the respiratory syncytial virus fusion protein in virus replication, syncytium formation and antigenicity.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches."}},"tag":"EXP"},{"id":417,"details":{"paperId":"09aa1d7f46fd7bae017e35e6cd727b14da4e4d95","externalIds":{"MAG":"3035178128","DOI":"10.1164/rccm.201908-1567oc","CorpusId":"219724398","PubMed":"32543879"},"title":"Chemokine (C-X-C motif) Ligand 4 is a Restrictor of Respiratory Syncytial Virus Infection and an Indicator of Clinical Severity.","abstract":"RATIONALE\nRespiratory syncytial virus (RSV) is the leading cause of childhood respiratory infections worldwide; however, no vaccine is available and treatment options are limited. Identification of host factors pivotal to viral replication may inform the development of novel therapies, prophylaxes, or diagnosis.\n\n\nOBJECTIVES\nTo identify host factors involved in RSV replication and to evaluate their potential for disease management.\n\n\nMETHODS\nA gain-of-function was performed based on a genome-wide human cDNA library screen for host factors involved in RSV replication. The antiviral mechanism of Chemokine (C-X-C motif) Ligand 4 (CXCL4) was analyzed. Its clinical role was evaluated via nasopharyngeal aspirates (NPA) and plasma samples from RSV-infected patients with different disease severities.\n\n\nMEASUREMENTS AND MAIN RESULTS\nForty-nine host factors restricting RSV replication were identified by gain-of-function screening, with CXCL4 showing the strongest antiviral effect, which was secretion-dependent. CXCL4 blocked viral attachment through binding to the RSV main receptor heparan sulfate, instead of interacting with RSV surface proteins. Intranasal pretreatment with CXCL4 alleviated inflammation in RSV-infected mice, as shown by decreased levels of tumor necrosis factor and viral load in bronchoalveolar lavage fluid samples as well as by viral N protein histological staining in lungs. Compared to non-RSV infections, RSV infections induced elevated CXCL4 concentrations both in plasma and airway samples from mice and pediatric patients. The airway CXCL4 level was correlated with viral load and disease severity in patients (P<0.001).\n\n\nCONCLUSIONS\nOur results suggest that CXCL4 is an RSV restriction factor which can block viral entry and serve as an indicator of clinical severity in RSV infections.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results suggest that CXCL4 is an RSV restriction factor which can block viral entry and serve as an indicator of clinical severity in RSV infections."}},"tag":"EXP"},{"id":94,"details":{"paperId":"756240bcb65111857821f9ef9bcdd7e5327b5de0","externalIds":{"MAG":"2737347025","DOI":"10.1016/j.virol.2017.07.024","CorpusId":"29493280","PubMed":"28750327"},"title":"Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A critical role of cholesterol in maintaining IAV and RSV membrane structure that is essential for sustaining viral stability and infectivity is indicated."}},"tag":"EXP"},{"id":327,"details":{"paperId":"851a284904586aa5fda8d09512a740326318b595","externalIds":{"MAG":"2046738822","DOI":"10.1128/JVI.06274-11","CorpusId":"206818816","PubMed":"22090136"},"title":"Cholesterol-Rich Microdomains as Docking Platforms for Respiratory Syncytial Virus in Normal Human Bronchial Epithelial Cells","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV entry appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process."}},"tag":"EXP"},{"id":90,"details":{"paperId":"396101173c1abf9750854f7b1599a67a777fd721","externalIds":{"MAG":"2112261869","DOI":"10.1016/j.virol.2011.10.029","CorpusId":"25037412","PubMed":"22088217"},"title":"Cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibro Blasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles."}},"tag":"EXP"},{"id":160,"details":{"paperId":"a46fa033f035541475d49665a47a2a963541f29c","externalIds":{"MAG":"2060028723","DOI":"10.1073/pnas.151098198","CorpusId":"11805981","PubMed":"11493675"},"title":"Cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion","abstract":"Preparations of purified full-length fusion (F) protein of human respiratory syncytial virus (HRSV) expressed in recombinant vaccinia-F infected cells, or of an anchorless mutant (FTM−) lacking the C-terminal 50 amino acids secreted from vaccinia-FTM−-infected cells contain a minor polypeptide that is an intermediate product of proteolytic processing of the F protein precursor F0. N-terminal sequencing of the intermediate demonstrated that it is generated by cleavage at a furin-motif, residues 106–109 of the F sequence. By contrast, the F1 N terminus derives from cleavage at residue 137 of F0 which is also C-terminal to a furin recognition site at residues 131–136. Site-directed mutagenesis indicates that processing of F0 protein involves independent cleavage at both sites. Both cleavages are required for the F protein to be active in membrane fusion as judged by syncytia formation, and they allow changes in F structure from cone- to lollipop-shaped spikes and the formation of rosettes by anchorless F.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Site-directed mutagenesis indicates that processing of F0 protein involves independent cleavage at both sites, and both cleavages are required for the F protein to be active in membrane fusion as judged by syncytia formation."}},"tag":"EXP"},{"id":114,"details":{"paperId":"f442e5c2395109a46b5fe50690d5a0142694451f","externalIds":{"MAG":"2038719395","DOI":"10.1016/S0168-1702(00)00149-0","CorpusId":"775924","PubMed":"10930660"},"title":"Cleavage of the respiratory syncytial virus fusion protein is required for its surface expression: role of furin.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The RSV F(0) protein could not be detected on the surface of cells, in which F protein activation was inhibited, and RSV particles did not appear to be released from these cells, suggesting that in contrast to the F proteins of most other paramyxoviruses, it is very inefficient in reaching the cellsurface or is unable to reach the cell surface and therefore cannot be incorporated into virus particles."}},"tag":"EXP"},{"id":120,"details":{"paperId":"b041b21dc917478dc8e7ee70492591db9959a032","externalIds":{"MAG":"1107047667","DOI":"10.1021/acs.biochem.5b00615","CorpusId":"141754","PubMed":"26237467"},"title":"Conformational Heterogeneity Determined by Folding and Oligomer Assembly Routes of the Interferon Response Inhibitor NS1 Protein, Unique to Human Respiratory Syncytial Virus.","abstract":"The nonstructural NS1 protein is an essential virulence factor of the human respiratory syncytial virus, with a predominant role in the inhibition of the host antiviral innate immune response. This inhibition is mediated by multiple protein-protein interactions and involves the formation of large oligomeric complexes. There is neither a structure nor sequence or functional homologues of this protein, which points to a distinctive mechanism for blocking the interferon response among viruses. The NS1 native monomer follows a simple unfolding kinetics via a nativelike transition state ensemble, with a half-life of 45 min, in agreement with a highly stable core structure at equilibrium. Refolding is a complex process that involves several slowly interconverting species compatible with proline isomerization. However, an ultrafast folding event with a half-life of 0.2 ms is indicative of a highly folding compatible species within the unfolded state ensemble. On the other hand, the oligomeric assembly route from the native monomer, which does not involve unfolding, shows a monodisperse and irreversible end-point species triggered by a mild temperature change, with half-lives of 160 and 26 min at 37 and 47 °C, respectively, and at a low protein concentration (10 μM). A large secondary structure change into β-sheet structure and the formation of a dimeric nucleus precede polymerization by the sequential addition of monomers at the surprisingly low rate of one monomer every 34 s. The polymerization phase is followed by the binding to thioflavin-T indicative of amyloid-like, albeit soluble, repetitive β-sheet quaternary structure. The overall process is reversible only up until ~8 min, a time window in which most of the secondary structure change takes place. NS1's multiple binding activities must be accommodated in a few binding interfaces at most, something to be considered remarkable given its small size (15 kDa). Thus, conformational heterogeneity, and in particular oligomer formation, may provide a means of expand its binding repertoire. These equilibria will be determined by variables such as macromolecular crowding, protein-protein interactions, expression levels, turnover, or specific subcellular localization. The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu. The implications of NS1 oligomers in the viral life cycle and the inhibition of host innate immune response remain to be determined.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu."}},"tag":"EXP"},{"id":164,"details":{"paperId":"24808802fc11d0988e41724bba7fab44bbe90001","externalIds":{"MAG":"2181932001","DOI":"10.1073/PNAS.80.5.1280","CorpusId":"42227870","PubMed":"6572388"},"title":"Construction and characterization of cDNA clones for four respiratory syncytial viral genes.","abstract":"Cytoplasmic poly(A)-containing RNA from respiratory syncytial virus-infected cells was used as a template to synthesize oligo(dT)-primed cDNAs. Discrete size classes of single-stranded cDNAs, resolved by alkali agarose gel electrophoresis, were used separately to construct double-stranded cDNAs that were subsequently inserted into the plasmid vector pBR322 at the Pst I site by means of oligo(dG)oligo(dC) tailing. After transfection of Escherichia coli, recombinant plasmids were screened mostly by serial rounds of hybrid selection of mRNAs from virus-infected cells and subsequent in vitro translation of the selected mRNAs. Comparative peptide mapping of the translation products with those of authentic virion proteins served to establish the viral origin of the cDNA recombinants. In this manner, four distinct classes of recombinant plasmids were identified. These encode sequences corresponding to those of respiratory syncytial virus nucleocapsid protein, matrix protein, phosphoprotein, and a nonstructural protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Recombinant plasmids encode sequences corresponding to those of respiratory syncytial virus nucleocapsid protein, matrix protein, phosphoprotein, and a nonstructural protein that serve to establish the viral origin of the cDNA recombinants."}},"tag":"EXP"},{"id":481,"details":{"paperId":"571db77ca301fee3cf77ac4fe8417dc4630cf8b8","externalIds":{"PubMedCentral":"6431622","MAG":"2922031945","DOI":"10.3389/fimmu.2019.00452","CorpusId":"80628545","PubMed":"30936869"},"title":"Contribution of Cytokines to Tissue Damage During Human Respiratory Syncytial Virus Infection","abstract":"The human respiratory syncytial virus (hRSV) remains one of the leading pathogens causing acute respiratory tract infections (ARTIs) in children younger than 2 years old, worldwide. Hospitalizations during the winter season due to hRSV-induced bronchiolitis and pneumonia increase every year. Despite this, there are no available vaccines to mitigate the health and economic burden caused by hRSV infection. The pathology caused by hRSV induces significant damage to the pulmonary epithelium, due to an excessive inflammatory response at the airways. Cytokines are considered essential players for the establishment and modulation of the immune and inflammatory responses, which can either be beneficial or harmful for the host. The deleterious effect observed upon hRSV infection is mainly due to tissue damage caused by immune cells recruited to the site of infection. This cellular recruitment takes place due to an altered profile of cytokines secreted by epithelial cells. As a result of inflammatory cell recruitment, the amounts of cytokines, such as IL-1, IL-6, IL-10, and CCL5 are further increased, while IL-10 and IFN-γ are decreased. However, additional studies are required to elicit the mediators directly associated with hRSV damage entirely. In addition to the detrimental induction of inflammatory mediators in the respiratory tract caused by hRSV, reports indicating alterations in the central nervous system (CNS) have been published. Indeed, elevated levels of IL-6, IL-8 (CXCL8), CCL2, and CCL4 have been reported in cerebrospinal fluid from patients with severe bronchiolitis and hRSV-associated encephalopathy. In this review article, we provide an in-depth analysis of the role of cytokines secreted upon hRSV infection and their potentially harmful contribution to tissue damage of the respiratory tract and the CNS.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"An in-depth analysis of the role of cytokines secreted upon hRSV infection and their potentially harmful contribution to tissue damage of the respiratory tract and the CNS is provided."}},"tag":"EXP"},{"id":421,"details":{"paperId":"033e8df00c8523ec434c593360bdc78b4d1bfc85","externalIds":{"PubMedCentral":"1540417","MAG":"1608890092","DOI":"10.1186/1743-422X-3-34","CorpusId":"16127684","PubMed":"16723026"},"title":"Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The mature F protein of all known isolates of human respiratory syncytial virus (HRSV) contains fifteen absolutely conserved cysteine (C) residues that are highly conserved among the F proteins of other pneumoviruses as well as the paramyxovirus."}},"tag":"EXP"},{"id":20,"details":{"paperId":"6a2fd13b1ab77c53d12d9ae2f0d4906c58e23e0e","externalIds":{"MAG":"2030577907","DOI":"10.1006/VIRO.2001.1138","CorpusId":"22914381","PubMed":"11689051"},"title":"Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo.","abstract":"The surface glycoproteins of viruses can play important roles in viral attachment, entry, and morphogenesis. Here, we investigated the role of the attachment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or express either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less efficiently and did not form distinct plaques in HEp-2 cells. This defect was primarily at the level of the initiation of infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the respiratory tract of mice was very highly restricted, indicating that G is important in vivo. Although the G protein expressed by the sG virus was confirmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains. Amino acids 184-198 have been identified as the major heparin-binding domain of the G protein and were implicated in mediating binding to cells [S. A. Feldman et al., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also appeared to be important for infection in vitro by direct clinical isolates of RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensitivity to neutralization in vitro by incubation with soluble heparin, did not reduce its efficiency of growth in vitro, and resulted in only a modest reduction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and is not a critical functional domain.","publicationTypes":["JournalArticle","Study"],"tldr":{"model":"tldr@v2.0.0","text":"The G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains."}},"tag":"EXP"},{"id":78,"details":{"paperId":"f4bf0f24ef88c317c68c6b8860f696b9bf711950","externalIds":{"MAG":"2084636380","DOI":"10.1016/J.VIROL.2004.10.010","CorpusId":"35612827","PubMed":"15582654"},"title":"Cooperativity of actin and microtubule elements during replication of respiratory syncytial virus.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that alteration of microtubule or actin function resulted in blocks at entry, formation of cell-associated virus, virus release, local cell-to-cell spread, and syncytium formation."}},"tag":"EXP"},{"id":468,"details":{"paperId":"1504d25c5b2860f72df403456d394f6b471c477a","externalIds":{"MAG":"2943343204","DOI":"10.1542/peds.2018-4064","CorpusId":"141443966","PubMed":"31040196"},"title":"Cost-effectiveness of Palivizumab for Respiratory Syncytial Virus: A Systematic Review","abstract":"In this systematic review, we investigate the cost-effectiveness of palivizumab prophylaxis for RSV stratified by setting and infant population subgroups relevant to health-policy decision-making. CONTEXT: Palivizumab prophylaxis is used as passive immunization for respiratory syncytial virus (RSV). However, because of its high cost, the value of this intervention is unclear. OBJECTIVE: To systematically review the cost-effectiveness of palivizumab prophylaxis compared with no prophylaxis in infants <24 months of age. DATA SOURCES: Medline, Embase, and Cochrane Library up to August 2018. STUDY SELECTION: Two reviewers independently screened results to include economic evaluations conducted between 2000 and 2018 from Organization for Economic Cooperation and Development countries. DATA EXTRACTION: Two reviewers independently extracted outcomes. Quality appraisal was completed by using the Joanna Briggs Institute checklist. Costs were adjusted to 2017 US dollars. RESULTS: We identified 28 economic evaluations (20 cost-utility analyses and 8 cost-effectiveness analyses); most were from the United States (n = 6) and Canada (n = 5). Study quality was high; 23 studies met >80% of the Joanna Briggs Institute criteria. Palivizumab prophylaxis ranged from a dominant strategy to having an incremental cost-effectiveness ratio of $2 526 203 per quality-adjusted life-year (QALY) depending on study perspective and targeted population. From the payer perspective, the incremental cost-effectiveness ratio for preterm infants (29–35 weeks’ gestational age) was between $5188 and $791 265 per QALY, with 90% of estimates <$50 000 per QALY. Influential parameters were RSV hospitalization reduction rates, palivizumab cost, and discount rate. LIMITATIONS: Model design heterogeneity, model parameters, and study settings were barriers to definitive conclusions on palivizumab’s economic value. CONCLUSIONS: Palivizumab as RSV prophylaxis was considered cost-effective in prematurely born infants, infants with lung complications, and infants from remote communities.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Palivizumab as RSV prophylaxis was considered cost-effective in prematurely born infants, infants with lung complications, and infants from remote communities."}},"tag":"DRUG"},{"id":178,"details":{"paperId":"9ed6748b17331298d6ee6e69a16a1a8dd22d22c2","externalIds":{"MAG":"1997077414","DOI":"10.1074/JBC.M502276200","CorpusId":"34555444","PubMed":"15784616"},"title":"Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences*","abstract":"We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered ∼150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Investigation of the mechanism of the translation of the second open reading frame of the respiratory syncytial virus M2 transcript showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur."}},"tag":"EXP"},{"id":282,"details":{"paperId":"678aea48df076b99f23511cbc8efcd6fdf3bd9d3","externalIds":{"MAG":"2076622648","DOI":"10.1128/JVI.00457-07","CorpusId":"11090431","PubMed":"17522208"},"title":"Coupled Translation of the Second Open Reading Frame of M2 mRNA Is Sequence Dependent and Differs Significantly within the Subfamily Pneumovirinae","abstract":"ABSTRACT Coupled translation, first described in the M2 gene of pneumovirus respiratory syncytial virus (RSV), is an alternative mechanism of translational initiation in which the ribosomes which translate the first (M2-1) open reading frame (ORF) move a short distance upstream after termination and reinitiate translation from a second (M2-2) overlapping ORF. Here, we show that the same mechanism occurs in two closely related viruses, avian pneumovirus (APV) and pneumonia virus of mice (PVM), although with markedly different efficiencies. To identify the reasons for the variation in efficiency of coupled expression between RSV and APV, we used chimeric M2-1 genes containing different lengths of the M2-1 ORF from each virus. An essential component allowing coupled expression in the chimeras was a segment from the RSV M2-1 coding region containing a high degree of secondary structure. Additional sequences at the 5′ end of the RSV M2-1 ORF also promoted coupled translation when the region with high levels of secondary structure was present. These data indicate that at least two distant parts of the mRNA transcript, together with a suitable overlapping region, are involved in the coupling process. Replacement of the last 102 nucleotides of the RSV M2-1 ORF with the equivalent APV sequence showed identical levels of coupled translation. Thus, the overlapping region can direct the ribosome back onto the start codon of the second ORF while the upstream coding sequence of the M2-1 ORF determines the levels of coupled expression.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data indicate that at least two distant parts of the mRNA transcript, together with a suitable overlapping region, are involved in the coupling process, and that the same mechanism occurs in two closely related viruses, avian pneumovirus (APV) and pneumonia virus of mice (PVM), although with markedly different efficiencies."}},"tag":"EXP"},{"id":141,"details":{"paperId":"3938d1cb719b11d538576f4780956183e689f281","externalIds":{"DOI":"10.1038/s41467-019-14246-3","CorpusId":"256635368"},"title":"Cryo-EM structure of the respiratory syncytial virus RNA polymerase","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminaldomain (PCTD) of a tetramer of P."}},"tag":"EXP"},{"id":265,"details":{"paperId":"be0aec5fe4515731e8a4c8ce50e9106e185809ca","externalIds":{"MAG":"2105403271","DOI":"10.1126/science.1177634","CorpusId":"5159732","PubMed":"19965480"},"title":"Crystal Structure of a Nucleocapsid-Like Nucleoprotein-RNA Complex of Respiratory Syncytial Virus","abstract":"RSV in 3D Respiratory syncytial virus (RSV) causes pneumonia and bronchiolitis in infants. RSV is an RNA virus in which the genomic RNA forms part of a nuclease-resistant helical ribonucleoprotein complex. Tawar et al. (p. 1279) now use x-ray and electron microscopy data to model the structure of this nucleocapsid complex and show how it can template RNA synthesis. The crystal structure shows RNA wrapped around a decameric ring of nucleocapsid protein. Combining this structure with electron microscopy data gives a model that shows how polymerase might read out the RNA bases without disassembling the nucleocapsid helix. In negative-strand RNA viruses, viral RNA wraps around a nucleocapsid helix with the bases accessible to the viral polymerase. The respiratory syncytial virus (RSV) is an important human pathogen, yet neither a vaccine nor effective therapies are available to treat infection. To help elucidate the replication mechanism of this RNA virus, we determined the three-dimensional (3D) crystal structure at 3.3 Å resolution of a decameric, annular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA. This complex mimics one turn of the viral helical nucleocapsid complex, which serves as template for viral RNA synthesis. The RNA wraps around the protein ring, with seven nucleotides contacting each N subunit, alternating rows of four and three stacked bases that are exposed and buried within a protein groove, respectively. Combined with electron microscopy data, this structure provides a detailed model for the RSV nucleocapsid, in which the bases are accessible for readout by the viral polymerase. Furthermore, the nucleoprotein structure highlights possible key sites for drug targeting.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The three-dimensional crystal structure at 3.3 Å resolution of a decameric, annular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA is determined, which mimics one turn of the viral helical nucleocapsid complex, which serves as template for viral RNA synthesis."}},"tag":"EXP"},{"id":158,"details":{"paperId":"c2cd6625db0c5128fba421d5ba4ec52dd4849c1a","externalIds":{"MAG":"2137876239","DOI":"10.1073/pnas.1317262111","CorpusId":"205265529","PubMed":"24434552"},"title":"Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation","abstract":"Significance Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children; however, no vaccine exists and current immunoprophylaxis regimes are both expensive and incompletely protective. We report the crystal structure of the HRSV M2-1 transcription factor that is essential for virus gene expression and thus growth. This structure reveals how M2-1 forms an extremely stable tetramer and has allowed us to pinpoint the location of critical regions that regulate M2-1 activity, providing insight into its function. This structure may represent a potent target for new antiviral compounds. The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This structure reveals how M2-1 forms an extremely stable tetramer and has allowed us to pinpoint the location of critical regions that regulate M1-1 activity, providing insight into its function and identifies surfaces critical for M 2-1 function that may be targeted by antiviral compounds."}},"tag":"EXP"},{"id":257,"details":{"paperId":"fb35f85798b536f7abf9d0c9bd1cd70e2f522f7d","externalIds":{"MAG":"1966822792","DOI":"10.1107/S1744309108031059","CorpusId":"25028571","PubMed":"18997331"},"title":"Crystallization and preliminary X-ray analysis of the human respiratory syncytial virus nucleocapsid protein.","abstract":"Human respiratory syncytial virus (HRSV) has a nonsegmented negative-stranded RNA genome which is encapsidated by the HRSV nucleocapsid protein (HRSVN) that is essential for viral replication. HRSV is a common cause of respiratory infection in infants, yet no effective antiviral drugs to combat it are available. Recent data from an experimental anti-HRSV compound, RSV-604, indicate that HRSVN could be the target site for drug action. Here, the expression, purification and preliminary data collection of decameric HRSVN as well as monomeric N-terminally truncated HRSVN mutants are reported. Two different crystal forms of full-length selenomethionine-labelled HRSVN were obtained that diffracted to 3.6 and approximately 5 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 133.6, b = 149.9, c = 255.1 A, and space group P2(1), with unit-cell parameters a = 175.1, b = 162.6, c = 242.8 A, beta = 90.1 degrees , respectively. For unlabelled HRSVN, only crystals belonging to space group P2(1) were obtained that diffracted to 3.6 A. A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN. Monomeric HRSVN generated by N-terminal truncation was designed to assist in structure determination by reducing the size of the asymmetric unit. Whilst such HRSVN mutants were monomeric in solution and crystallized in a different space group, the size of the asymmetric unit was not reduced.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A self-rotation function using data from the orthorhombic crystal form confirmed the presence of tenfold noncrystallographic symmetry, which is in agreement with a reported electron-microscopic reconstruction of HRSVN."}},"tag":"EXP"},{"id":7,"details":{"paperId":"947bf313d975773e19da9da74ee2a4641a89e7e3","externalIds":{"MAG":"1974451794","DOI":"10.1006/VIRO.1993.1366","CorpusId":"19111332","PubMed":"8317099"},"title":"Cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22K protein.","abstract":"Immunofluorescence staining and immunoelectron microscopy of respiratory syncytial (RS) virus-infected cells revealed the presence of cytoplasmic inclusions that were specifically labeled with monoclonal antibodies directed against the nucleoprotein (NP), the phosphoprotein (P), or the 22K protein. Transient expression of these three proteins with the vaccinia-T7 system, either individually or in different combinations, demonstrated that coexpression of NP and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in RS virus-infected cells. In addition, the 22K protein was also incorporated to the inclusions when coexpressed with both NP and P proteins. Immunobinding assays revealed the presence of NP-P and NP-22K complexes in extracts of RS virus-infected cells. These complexes were also detected in extracts of transfected cells that coexpressed the corresponding proteins. The implications of these results for the RS virus replicative cycle are discussed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Transient expression of these three proteins with the vaccinia-T7 system demonstrated that coexpression of NP and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in RS virus-infected cells."}},"tag":"EXP"},{"id":212,"details":{"paperId":"11b0d9df91bf120c52f4f8c58fb30606110a87c7","externalIds":{"MAG":"2131354424","DOI":"10.1099/0022-1317-68-9-2521","CorpusId":"32032687","PubMed":"3655746"},"title":"Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus.","abstract":"Two monospecific rabbit antisera to the G glycoprotein, one induced with purified G and the second with a recombinant vaccinia virus containing the gene for G, inhibit the attachment of purified [35S]methionine-labelled Long strain of respiratory syncytial virus to monolayers of HeLa cells. Attachment was not inhibited by monospecific rabbit antisera to glycoprotein F induced with either purified F or with a recombinant vaccinia virus containing the gene for F.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Two monospecific rabbit antisera to the G glycoprotein inhibit the attachment of purified [35S]methionine-labelled Long strain of respiratory syncytial virus to monolayers of HeLa cells."}},"tag":"EXP"},{"id":225,"details":{"paperId":"a5e489d996dd98a3db059aa4e5da5d6e4eecae6b","externalIds":{"MAG":"2132300794","DOI":"10.1099/0022-1317-80-8-2011","CorpusId":"2998122","PubMed":"10466798"},"title":"Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses.","abstract":"The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, having 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA also contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant sequence identity could be detected between the putative M2 ORF2 proteins of PVM, APV and the RS viruses. The expression of the M2 ORF2 proteins of the pneumoviruses was investigated by using monospecific antisera raised against GST fusion proteins. Western blot analysis demonstrated the presence of polypeptides encoded by M2 ORF2 of PVM and RS virus corresponding with those predicted by in vitro translation studies, but this was not the case for APV. The PVM polypeptide was present as three distinct products in vivo. The PVM and RS virus polypeptides were also detected in cells by immunofluorescence, which showed that both were present in the cytoplasm with a degree of localization in inclusion bodies. No APV M2 ORF2 protein could be detected in vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during infection and the potential implications of this are discussed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The nucleotide sequence of the M2 gene of pneumonia virus of mice showed that the gene encoded a protein of 176 amino acids with a predicted molecular mass of 20165 Da from a major ORF, which is smaller than the equivalent proteins encoded by human, bovine and ovine respiratory syncytial viruses."}},"tag":"EXP"},{"id":243,"details":{"paperId":"cb488445065f35484718fbc5b0452620fac5ce63","externalIds":{"MAG":"2165024752","DOI":"10.1099/VIR.0.80692-0","CorpusId":"37917653","PubMed":"15784905"},"title":"Determination of phosphorylated residues from human respiratory syncytial virus P protein that are dynamically dephosphorylated by cellular phosphatases: a possible role for serine 54.","abstract":"The 241 aa human respiratory synctyial virus (HRSV) Long strain P protein is phosphorylated at serines 116, 117 and/or 119, and 232. Phosphates added to these residues have slow turnover and can be detected in the absence of protein phosphatase inhibition. Inhibition of phosphatases PP1 and PP2A increases the level of phosphorylation at serines 116, 117 and/or 119, suggesting a more rapid turnover for phosphates added to these residues compared to that of S232. High-turnover phosphorylation is detected in the P-protein NH2-terminal region, mainly at S54 and, to a lesser extent, at S39, in the Long strain. When the P protein bears the T46I substitution (in the remaining HRSV strains), phosphates are added to S30, S39, S45 and S54. Phosphatase PP1 removes phosphate at residues in the central part of the P-protein molecule, whereas those in the NH2-terminal region are removed by phosphatase PP2A. The significance of the phosphorylation of the NH2-terminal region residues for some P-protein functions was studied. The results indicated that this modification is not essential for P-protein oligomerization or for its role in viral RNA synthesis. Nonetheless, dephosphorylation at S54 could facilitate P-M protein interactions that probably occur during the egress of viral particles.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results indicated that this modification is not essential for P-protein oligomerization or for its role in viral RNA synthesis, but dephosphorylation at S54 could facilitate P-M protein interactions that probably occur during the egress of viral particles."}},"tag":"EXP"},{"id":4,"details":{"paperId":"78f126f990fb1d2cdbaf16aff2e884586208fec8","externalIds":{"MAG":"2133771235","DOI":"10.1002/PRO.5560060619","CorpusId":"36291545","PubMed":"9194191"},"title":"Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry","abstract":"The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse‐phase high‐performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight (TOF) mass spectrometry (MS) corresponding to residues 152‐187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub‐digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys 173 and Cys 186 and between Cys 176 and Cys 182. Analysis of ions produced by post‐source decay of a peptic peptide during MALDI‐TOF‐MS revealed fragmentation of peptide bonds with minimal fission of an inter‐chain disulfide bond. Ions produced by this unprecedented MALDI‐induced post‐source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non‐glycosylated subdomain containing a “cystine noose.”","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Findings indicate that the ectodomain of the G protein has a non‐glycosylated subdomain containing a “cystine noose” that corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products."}},"tag":"EXP"},{"id":416,"details":{"paperId":"146dbeb0f1a6af4b1043728740909cc601b6c411","externalIds":{"MAG":"2973696106","DOI":"10.1159/000502674","CorpusId":"202688388","PubMed":"31533107"},"title":"Differences in Susceptibility of Human and Mouse Macrophage Cell Lines to Respiratory Syncytial Virus Infection","abstract":"Objectives: Differences have been observed in the susceptibility of macrophage cell lines to respiratory syncytial virus (RSV) infection. In this study, we evaluated whether the type of macrophage cell line and RSV strain used have an influence on the infectivity and production of progeny virus. Methods: Both human and murine macrophage-like cell lines were infected with different RSV strains, both lab strains as well as clinical isolates. The infection was evaluated after 24 and 72 h by immunofluorescence staining and microscopic analysis, and the production of new virus particles was determined by plaque assay. Results: Susceptibility of macrophages to RSV was influenced by the RSV strain used but was mostly dependent on the macrophage cell line. Numbers of infected cells and virus production were generally very low or absent in murine cell lines. In human cell lines, clear infection was observed associated with production of new virus particles. Conclusion: Differences in susceptibility of macrophage cell lines to RSV infection are primarily related to the species of origin of the cell line but are also influenced by the RSV strain.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Differences in susceptibility of macrophage cell lines to RSV infection are primarily related to the species of origin of the cell line but are also influenced by the RSV strain."}},"tag":"EXP"},{"id":322,"details":{"paperId":"97251f654718b81ee24ee1730b7046954aee96dd","externalIds":{"MAG":"2010124950","DOI":"10.1128/JVI.03500-14","CorpusId":"576244","PubMed":"25673702"},"title":"Dimerization of Matrix Protein Is Required for Budding of Respiratory Syncytial Virus","abstract":"ABSTRACT Respiratory syncytial virus (RSV) infects epithelial cells of the respiratory tract and is a major cause of bronchiolitis and pneumonia in children and the elderly. The virus assembles and buds through the plasma membrane, forming elongated membrane filaments, but details of how this happens remain obscure. Oligomerization of the matrix protein (M) is a key step in the process of assembly and infectious virus production. In addition, it was suggested to affect the conformation of the fusion protein, the major current target for RSV antivirals, in the mature virus. The structure and assembly of M are thus key parameters in the RSV antiviral development strategy. The structure of RSV M was previously published as a monomer. Other paramyxovirus M proteins have been shown to dimerize, and biochemical data suggest that RSV M also dimerizes. Here, using size exclusion chromatography-multiangle laser light scattering, we show that the protein is dimeric in solution. We also crystallized M in two crystal forms and show that it assembles into equivalent dimers in both lattices. Dimerization interface mutations destabilize the M dimer in vitro. To assess the biological relevance of dimerization, we used confocal imaging to show that dimerization interface mutants of M fail to assemble into viral filaments on the plasma membrane. Additionally, budding and release of virus-like particles are prevented in M mutants that fail to form filaments. Importantly, we show that M is biologically active as a dimer and that the switch from M dimers to higher-order oligomers triggers viral filament assembly and virus production. IMPORTANCE Human respiratory syncytial virus (RSV) is the most frequent cause of infantile bronchiolitis and pneumonia. The enormous burden of RSV makes it a major unmet target for a vaccine and antiviral drug therapy. Oligomerization of the matrix protein is a key step in the process of assembly and production of infectious virus, but the molecular mechanism of RSV assembly is still poorly understood. Here we show that the RSV matrix protein forms dimers in solution and in crystals; the dimer is essential for formation of higher-order oligomers. Destabilizing the dimer interface resulted in the loss of RSV filament formation and a lack of budding of virus-like particles. Importantly, our findings can potentially lead to new structure-based RSV inhibitors targeting the assembly process.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that the RSV matrix protein forms dimers in solution and in crystals; the dimer is essential for formation of higher-order oligomers, and can potentially lead to new structure-based RSV inhibitors targeting the assembly process."}},"tag":"EXP"},{"id":454,"details":{"paperId":"696398e7316a1195c4fd5bebce08c26a871e68c6","externalIds":{"PubMedCentral":"6023113","MAG":"2810988926","DOI":"10.1371/journal.ppat.1007015","CorpusId":"49586510","PubMed":"29953542"},"title":"Direct cell-to-cell transmission of respiratory viruses: The fast lanes","abstract":"Virus particles protect genomes from hostile environments within and outside the host, eventually delivering these genomes to target tissues to initiate infection. Complex processes requiring significant energy and time are necessary to assemble these virus particles, but only a small portion of released virus will successfully infect new target cells (Fig 1A). While the science of virology has developed based on the isolation and purification of viral particles, it is becoming increasingly clear that direct cell-to-cell transmission of viruses and/or viral components is also highly relevant [1,2]. Direct cell-to-cell spread of infections has several advantages. The first is efficiency: genomic cargo is delivered directly to cells rather than being randomly released into the environment. The second is speed: appropriation of cellular protein trafficking infrastructure allows faster spread within tissues. The third is barrier avoidance: intrinsic immunity and other barriers interfering with entry or post-entry steps in target cells can be bypassed. The fourth is humoral immunity evasion: limited exposure time to the extracellular space allows evasion of neutralizing antibodies.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Direct cell-to-cell spread of infections has several advantages: efficiency: genomic cargo is delivered directly to cells rather than being randomly released into the environment, speed: appropriation of cellular protein trafficking infrastructure allows faster spread within tissues, and barrier avoidance."}},"tag":"EXP"},{"id":61,"details":{"paperId":"4a03feb71209e7744cfc41c02b135a7f4b007d10","externalIds":{"PubMedCentral":"2896471","MAG":"2077441505","DOI":"10.1016/j.febslet.2010.05.006","CorpusId":"29975767","PubMed":"20471980"},"title":"Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"MINT‐7890784, MINT-7890776: SH (uniprot kb:P04852) and SH ( uniprotkb:P02452) bind (MI:0407) by electron microscopy by electron microscope."}},"tag":"EXP"},{"id":465,"details":{"paperId":"de14b745b26b7b4ced3b6f6ae665885140f8f34b","externalIds":{"PubMedCentral":"9255747","DOI":"10.1371/journal.ppat.1010451","CorpusId":"247599919","PubMed":"35731802"},"title":"Distinctive features of the respiratory syncytial virus priming loop compared to other non-segmented negative strand RNA viruses","abstract":"De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation. Author Summary RSV has a significant impact on human health. It is the major cause of respiratory disease in infants and exerts a significant toll on the elderly and immunocompromised. RSV is a member of the Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs). Like other viruses in this order, RSV encodes an RNA dependent RNA polymerase, which is responsible for transcribing and replicating the viral genome. Due to its essential role during the viral replication cycle, the polymerase is a promising candidate target for antiviral inhibitors and so a greater understanding of the mechanistic basis of its activities could aid antiviral drug development. In this study, we identified an amino acid residue within the RSV polymerase that appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication. However, the corresponding residue in a different nsNSV polymerase does not appear to play a similar role. This work reveals a key feature of the RSV polymerase but identifies differences with the polymerases of other related viruses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"An amino acid residue within the RSV polymerase appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication, however, the corresponding residue in a different nsNSV polymerases does not appear to play a similar role."}},"tag":"EXP"},{"id":112,"details":{"paperId":"6b58d9694fd8e84927770c17f407a3b96adcf40a","externalIds":{"MAG":"2068473963","DOI":"10.1016/S0042-6822(02)00016-8","CorpusId":"20958788","PubMed":"12642099"},"title":"Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus."}},"tag":"EXP"},{"id":131,"details":{"paperId":"6e57442979c4e288a8af4ba124f62a19090cd44d","externalIds":{"MAG":"45460210","DOI":"10.1023/A:1008188527858","CorpusId":"2722481","PubMed":"10949949"},"title":"Domains of Human Respiratory Syncytial Virus P Protein Essential for Homodimerization and for Binding to N and NS1 Protein","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The two-hybrid technique was used to test for binding among human respiratory syncytial virus proteins involved in the control of viral replication and the expected positive interactions for the nucleoprotein (N) with itself and the phosphop protein (P)."}},"tag":"EXP"},{"id":449,"details":{"paperId":"0c6fe5cd20b037e1365f54c12dc2f633d8dc5404","externalIds":{"PubMedCentral":"4859522","MAG":"2347142754","DOI":"10.1371/journal.ppat.1005622","CorpusId":"2714347","PubMed":"27152417"},"title":"EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression","abstract":"Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2–20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore, we hypothesized that the RSV 2–20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2–20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2–20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2–20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EG FR is a potential target for RSV disease."}},"tag":"EXP"},{"id":485,"details":{"paperId":"932b9c91af30f4b3803b82289dd4e94849bfe3b4","externalIds":{"PubMedCentral":"7884774","DOI":"10.3389/fmolb.2020.609732","CorpusId":"231733409","PubMed":"33604354"},"title":"ELAC2, an Enzyme for tRNA Maturation, Plays a Role in the Cleavage of a Mature tRNA to Produce a tRNA-Derived RNA Fragment During Respiratory Syncytial Virus Infection","abstract":"Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in young children. However, effective treatment against RSV is unavailable. tRNA-derived RNA fragments (tRFs) are a recently discovered family of non-coding RNAs. We made an early observation that RSV infection causes significant induction of tRFs, which are mainly derived from the 5’-end of mature tRNAs (tRF5). However, their functions and biogenesis mechanism are not fully understood. Herein, we identified an enzyme responsible for the induction of a functional tRF5 derived from tRNA-Gln-CTG (tRF5-GlnCTG). We found that tRF5-GlnCTG promotes RSV replication and its induction, assessed by Northern blot and a new qRT-PCR-based method, is regulated by ribonuclease ELAC2. ELAC2-mediated tRF5 induction has never been reported. We also found that ELAC2 is associated with RSV N and NS1 proteins. Given the fact that tRF5-GlnCTG plays a role in RSV replication, the identification of ELAC2 being responsible for tRF5-GlnCTG induction could provide new insights into therapeutic strategy development against RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that tRF5-GlnCTG promotes RSV replication and its induction, assessed by Northern blot and a new qRT-PCR-based method, is regulated by ribonuclease ELAC2."}},"tag":"EXP"},{"id":110,"details":{"paperId":"77d91b3894a01c7feff55e89a1568b27975d92c5","externalIds":{"MAG":"1974829806","DOI":"10.1016/S0014-5793(04)00002-X","CorpusId":"40889186","PubMed":"14960303"},"title":"ERK‐1/2 activity is required for efficient RSV infection","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that the activation of the ERK‐1/2 is required in RSV‐induced early gene expression, and inhibition of ERK pathways significantly decreases RSV infection of these cells compared to controls."}},"tag":"EXP"},{"id":22,"details":{"paperId":"15d5d1142ff283d38b943f5b29304624114bd18b","externalIds":{"MAG":"2045515452","DOI":"10.1006/VIRO.2002.1497","CorpusId":"12668295","PubMed":"12127793"},"title":"Effect of proteolytic processing at two distinct sites on shape and aggregation of an anchorless fusion protein of human respiratory syncytial virus and fate of the intervening segment.","abstract":"We have examined the consequences of cleaving the fusion glycoprotein (F) of human respiratory syncytial virus (HRSV) at two distinct furin-recognition sites. Purified anchorless F is a mixture of unaggregated cone-shaped molecules and rosettes of lollipop-shaped spikes. The unaggregated molecules contain a proportion of uncleaved F0 and an intermediate, F(delta1-109), cleaved only at site I, residues 106-109. Inhibition of cleavage at site I, by two amino acid changes (R108N/R109N), reduces the proportion of aggregated molecules with a concomitant increase in the amount of unprocessed F0. Inhibition of cleavage at site II, residues 131-136, by deletion of four amino acids (delta131-134), abrogates aggregation of anchorless F and all molecules are seen as individual cone-shaped rods. In vitro cleavage of anchorless F, or mutant delta131-134, with trypsin at 4, 20, or 37 degrees C, under conditions in which cleavage at site II is complete in all molecules, leads to their aggregation in rosettes of lollipop-shaped spikes. Thus, cleavage at site II is required for the structural changes in anchorless F that lead to changes in shape and to aggregation. The segment between sites I and II, residues 110-136, is not associated with anchorless F in the supernatant of infected cell cultures, indicating that it is released from the processed protein when cleavage at sites I and II is completed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In vitro cleavage of anchorless F, or mutant delta131-134, with trypsin at 4, 20, or 37 degrees C, under conditions in which cleavage at site II is complete in all molecules, leads to their aggregation in rosettes of lollipop-shaped spikes."}},"tag":"EXP"},{"id":17,"details":{"paperId":"9d218ac3caa5a55c74925099ae0cff97df7d4e75","externalIds":{"MAG":"2033882116","DOI":"10.1006/VIRO.2000.0279","CorpusId":"20385842","PubMed":"10814577"},"title":"Electron microscopy of the human respiratory syncytial virus fusion protein and complexes that it forms with monoclonal antibodies.","abstract":"Full-length fusion (F) glycoprotein of human respiratory syncytial virus (HRSV) and a truncated anchorless mutant lacking the C-terminal 50 amino acids were expressed from vaccinia recombinants and purified by immunoaffinity chromatography and sucrose gradient centrifugation. Electron microscopy of full-length F protein in the absence of detergents revealed micelles, (i.e., rosettes) containing two distinct types of protein rods, one cone-shaped and the other lollipop-shaped. Analysis of membrane anchorless F molecules indicated that they were similar to the cone-shaped rods and that rosettes, which they formed on storage, were made up of lollipop-shaped rods. The two forms of F protein may represent different structures that the molecule may adopt before and after activation for its role in membrane fusion. Studies of complexes of these structures with monoclonal antibodies of known specificity provide information on the three-dimensional organization of antigenic sites on the F protein and confirm the oligomeric structure, possibly trimeric, of both full-length F and membrane anchorless F.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Electron microscopy of full-length F protein in the absence of detergents revealed micelles, (i.e., rosettes) containing two distinct types of protein rods, one cone-shaped and the other lollipop-shaped, which may represent different structures that the molecule may adopt before and after activation for its role in membrane fusion."}},"tag":"EXP"},{"id":89,"details":{"paperId":"bb755c3a528e905228cbb4d854f87f996ac3c616","externalIds":{"MAG":"2042875479","DOI":"10.1016/j.virol.2009.10.040","CorpusId":"205644172","PubMed":"19922971"},"title":"Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy."}},"tag":"EXP"},{"id":135,"details":{"paperId":"07b9da62cc586fadb2b38e21f67171f1a57eacae","externalIds":{"MAG":"2415209296","DOI":"10.1038/mt.2015.161","CorpusId":"45526616","PubMed":"26442802"},"title":"Emerging Roles of tRNA-derived Fragments in Viral Infections: The Case of Respiratory Syncytial Virus.","abstract":null,"publicationTypes":["LettersAndComments","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that tRFs may be targets for the development of new drugs for the treatment of respiratory tract infections caused by RSV because they enhance viral replication and counteract the host immune response by modulating gene expression at the posttranscriptional level."}},"tag":"EXP"},{"id":233,"details":{"paperId":"0312938a953ca37f851263eeb37b86728b46dd87","externalIds":{"MAG":"2951114697","DOI":"10.1099/jgv.0.001287","CorpusId":"184485344","PubMed":"31184573"},"title":"Evaluation of the role of respiratory syncytial virus surface glycoproteins F and G on viral stability and replication: implications for future vaccine design.","abstract":"Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability."}},"tag":"EXP"},{"id":255,"details":{"paperId":"52703b37c09a5ff4bd0d6efa73e8d30e53617107","externalIds":{"DOI":"10.1101/2022.12.18.520517","CorpusId":"254879210"},"title":"Evidence for a functional interaction between the respiratory syncytial virus fusion and attachment proteins in the envelope of infectious virus particles","abstract":"We have examined the interaction between the respiratory syncytial virus (RSV) F and G proteins on the surface of infected cells during multiple cycle infection using a low multiplicity of infection (MOI) model, and on the surface of virus particles that were isolated from infected cells. A combination of the proximity ligation assay (PLA) and confocal microscopy was used to demonstrate the interaction between the F and G proteins within the virus filaments on infected cells. Co-precipitation of the F and G proteins was confirmed using detergent extracts prepared from infected cells and in detergent extracts prepared from purified virus particles. The influence of the G protein in mediating virus spread in the low MOI model was further examined using the recombinant virus isolates rg224RSV (that expresses all virus proteins) and rg224RSV-ΔG (which does not express the G protein). While cells could be initially infected by both viruses, the rg224RSV-ΔG virus exhibited severely impaired localised virus transmission in the multiple cycle infection assay. Collectively these data provide evidence that the F and G proteins interact within the envelope of RSV particles, and suggests that this interaction may promote virus transmission. The interaction between these proteins in a single protein complex represents a potential new target for the development of antivirus strategies and in the development of RSV vaccine candidates.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Evidence that the F and G proteins interact within the envelope of RSV particles, and suggests that this interaction may promote virus transmission, is provided."}},"tag":"EXP"},{"id":80,"details":{"paperId":"c40f532254611d9ed9d03dbe53e3a2280ca5f6ea","externalIds":{"MAG":"2065775449","DOI":"10.1016/J.VIROL.2005.05.004","CorpusId":"22340006","PubMed":"15936795"},"title":"Evidence for an association between heat shock protein 70 and the respiratory syncytial virus polymerase complex within lipid-raft membranes during virus infection.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Analysis of the HSP70 content in lipid-raft membranes from mock- and virus-infected cells revealed an increase in the levels of raft-associated HSP 70 during virus infection, suggesting that RSV may induce cellular changes which allow the recruitment of specific host-cell factors, via lipid-RAFT membranes, to the polymerase complex."}},"tag":"EXP"},{"id":84,"details":{"paperId":"6057af9c0cfca026adbf72be567308ce0a985d3b","externalIds":{"MAG":"2024952314","DOI":"10.1016/J.VIROL.2006.01.023","CorpusId":"40308359","PubMed":"16513154"},"title":"Evidence that maturation of the N-linked glycans of the respiratory syncytial virus (RSV) glycoproteins is required for virus-mediated cell fusion: The effect of alpha-mannosidase inhibitors on RSV infectivity.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity."}},"tag":"EXP"},{"id":88,"details":{"paperId":"8027a0d4b57b75412e588b31a41c6c3488f8dca6","externalIds":{"MAG":"2039217524","DOI":"10.1016/j.virol.2008.12.017","CorpusId":"21561572","PubMed":"19178924"},"title":"Evidence that selective changes in the lipid composition of raft-membranes occur during respiratory syncytial virus infection.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The analysis suggests that RSV infection induces specific changes in the composition of raft-associated lipids, and that these changes play an important role in virus maturation."}},"tag":"EXP"},{"id":390,"details":{"paperId":"b81e19fb52fca5b0895e0a6c4cf6c62fe922b35b","externalIds":{"MAG":"2149493383","DOI":"10.1128/JVI.79.17.11311-11322.2005","CorpusId":"39053179","PubMed":"16103183"},"title":"Evidence that the Respiratory Syncytial Virus Polymerase Is Recruited to Nucleotides 1 to 11 at the 3′ End of the Nucleocapsid and Can Scan To Access Internal Signals","abstract":"ABSTRACT The 3′-terminal end of the respiratory syncytial virus genomic RNA contains a 44-nucleotide leader (Le) region adjoining the gene start signal of the first gene. Previous mapping studies demonstrated that there is a promoter located at the 3′ end of Le, which can signal initiation of antigenome synthesis. The aim of this study was to investigate the role of the 3′ terminus of the RNA template in (i) promoter recognition and (ii) determining the initiation site for antigenome synthesis. A panel of minigenomes containing additional sequence at the 3′ end of the Le were analyzed for their ability to direct antigenome and mRNA synthesis. Minigenomes containing heterologous extensions of 6 nucleotides or more were unable to support efficient RNA synthesis. However, the activity of a minigenome with a 56-nucleotide extension could be restored by insertion of Le nucleotides 1 to 11 or 1 to 13 at the 3′ end, indicating that these nucleotides, in conjunction with the 3′ terminus, are sufficient to recruit polymerase to the template. Northern blot and 5′ rapid amplification of cDNA ends analysis of antigenome RNA indicated that antigenome initiation occurred at the first position of Le, irrespective of the terminal extension. This finding demonstrates that the 3′ terminus of the RNA is not necessary for determining the antigenome initiation site. Data are presented which suggest that following recruitment to a promoter at the 3′ end of Le, the polymerase is able to scan and respond to a promoter signal embedded within the RNA template.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Northern blot and 5′ rapid amplification of cDNA ends analysis of antigenome RNA indicated that antigenome initiation occurred at the first position of Le, irrespective of the terminal extension, demonstrating that the 3′ terminus of the RNA is not necessary for determining the antigenomes initiation site."}},"tag":"EXP"},{"id":31,"details":{"paperId":"8d54bd799e84e37a251be3e53054043c3d3776af","externalIds":{"MAG":"2065682986","DOI":"10.1007/BF01313961","CorpusId":"2365102","PubMed":"2774976"},"title":"Evidence that the fusion protein of respiratory syncytial virus exists as a dimer in its native form","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In situ crosslinking studies demonstrated that the fusion protein of RSV exists as a dimer in its native form on the surface of infected cells and suggested that F1 subunits may play a role in the dimerization of the fusionprotein."}},"tag":"EXP"},{"id":157,"details":{"paperId":"47fccdaaf132e7cec766ba7355999b9f969e55e7","externalIds":{"MAG":"2102470405","DOI":"10.1073/pnas.0913065107","CorpusId":"205247229","PubMed":"20479224"},"title":"Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion","abstract":"RNA virus polymerases must initiate replicative RNA synthesis with extremely high accuracy to maintain their genome termini and to avoid generating defective genomes. For the single-stranded negative-sense RNA viruses, it is not known how this accuracy is achieved. To investigate this question, mutations were introduced into the 3′ terminal base of a respiratory syncytial virus (RSV) template, and the RNA products were examined to determine the impact of the mutation. To perform the assay, RNA replication was reconstituted using a modified minireplicon system in which replication was limited to a single step. Importantly, this system allowed analysis of RSV RNA generated intracellularly, but from a defined template that was not subject to selection by replication. Sequence analysis of RNA products generated from templates containing 1U-C and 1U-A substitutions showed that, in both cases, replication products were initiated with a nontemplated, WT A residue, rather than a templated G or U residue, indicating that the polymerase selects the terminal NTP independently of the template. Examination of a template in which the position 1 nucleotide was deleted supported these findings. This mutant directed efficient replication at ∼60% of WT levels, and its product was found to be initiated at the WT position (−1 relative to the template) with a WT A residue. These findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Findings show that the RSV replicase selects ATP and initiates at the correct position, independently of the first nucleotide of the template, suggesting a mechanism by which highly accurate replication initiation is achieved."}},"tag":"EXP"},{"id":76,"details":{"paperId":"a2e418e2b9bc8b601f52b4493ac4fbfe902cfa96","externalIds":{"MAG":"2042627013","DOI":"10.1016/J.VIROL.2004.09.034","CorpusId":"32992353","PubMed":"15527841"},"title":"Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Evidence is provided that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells and an association between these proteins and the raft-lipid, GM1."}},"tag":"EXP"},{"id":52,"details":{"paperId":"b2369969d713dead0e20c801a19c5b95aef09947","externalIds":{"MAG":"2081677805","DOI":"10.1016/0168-1702(96)01327-5","CorpusId":"24423869","PubMed":"8864205"},"title":"Expression and characterisation of the NS1 and NS2 proteins of respiratory syncytial virus.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results indicate that the NS1 and NS2 proteins have distinct roles in the viral life cycle and could be co-precipitated by a monoclonal antibody to M protein."}},"tag":"EXP"},{"id":45,"details":{"paperId":"7166ab09076137bf4b4c310eba8a575025c6f58b","externalIds":{"MAG":"2028166387","DOI":"10.1016/0042-6822(87)90485-5","CorpusId":"8295452","PubMed":"3113070"},"title":"Expression and glycosylation of the respiratory syncytial virus G protein in Saccharomyces cerevisiae.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that yeast can recognize the internal signal sequence of RSV G protein and add glycosyl groups to the polypeptide in the endoplasmic reticulum and evidence is presented for both N- and O-linked Glycosylation of the virus glycoprotein."}},"tag":"EXP"},{"id":48,"details":{"paperId":"009f608501a7ad52712d5efb8451dcb2802665fa","externalIds":{"MAG":"2037611465","DOI":"10.1016/0042-6822(91)90863-7","CorpusId":"29852671","PubMed":"1714666"},"title":"Expression of the F glycoprotein gene from human respiratory syncytial virus in Escherichia coli: mapping of a fusion inhibiting epitope.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A cDNA copy of the gene encoding the entire amino acid sequence of the fusion (F) protein of human respiratory syncytial virus was inserted into a bacterial expression vector containing the lambda PR promoter, resulting in severe inhibition of bacterial growth and deletion of the DNA sequences encoding the F signal peptide."}},"tag":"EXP"},{"id":194,"details":{"paperId":"0acbd8623edb9675e1cd6f39891a90cad8a5231b","externalIds":{"MAG":"2063298404","DOI":"10.1093/emboj/19.11.2681","CorpusId":"6672154","PubMed":"10835365"},"title":"Expression of the ORF‐2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination‐dependent reinitiation mechanism","abstract":"Translation of the open reading frame 2 (ORF‐2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located upstream of the termination codon for the first ORF. Replacement of ORF‐2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORF‐2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF‐1 protein plays a crucial role in directing translation of ORF‐2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5′ to the point of translation termination.","publicationTypes":["JournalArticle","Study"],"tldr":{"model":"tldr@v2.0.0","text":"Replacement of ORF‐2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORf‐2 expression both in vitro and in vivo."}},"tag":"EXP"},{"id":208,"details":{"paperId":"03338e16ff4b5f8e59f93c7b982976b9254c3fd2","externalIds":{"MAG":"2171315624","DOI":"10.1099/0022-1317-68-4-1217","CorpusId":"24572212","PubMed":"3553424"},"title":"Expression of the respiratory syncytial virus 22K protein on the surface of infected HeLa cells.","abstract":"Immunofluorescent staining of unfixed respiratory syncytial virus-infected HeLa cells with monoclonal antibodies (MAbs) demonstrated that the 22K protein is expressed on the cell membrane along with the fusion (F) protein and large glycoprotein (G). All three proteins were detected in the cytoplasm at 17 h post-infection and in the case of the F and G proteins this coincided with their appearance on the cell surface. However, the 22K protein could not be detected on the surface until approximately 16 h after its detection in the cytoplasm, when cytopathic effect was extensive. No evidence for the surface expression of the phosphoprotein (P), matrix (M) or nucleocapsid (N) proteins was found. Trypsin treatment of infected cells prior to unfixed immunofluorescent staining and Western blot analysis indicated that, unlike the G protein, the quantity of 22K protein detected on the cell surface constituted only a small proportion of the total present in the cell. A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A comparison of the patterns of immunofluorescent staining produced by MAbs on acetone-fixed infected cells suggested that the N, P and 22K proteins, but not the M protein, may be associated with the same intracellular structures."}},"tag":"EXP"},{"id":423,"details":{"paperId":"3a544cd1b4cf826075db2a27b0dfde28dc808596","externalIds":{"PubMedCentral":"1751022","MAG":"1652049651","DOI":"10.1186/cc4984","CorpusId":"4260714","PubMed":"16859512"},"title":"Extrapulmonary manifestations of severe respiratory syncytial virus infection – a systematic review","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In infants with severe RSV infection cardiac rhythm, blood pressure and serum sodium need to be monitored and supportive treatment including fluid management adjusted accordingly, indicating a systemic dissemination of RSV during severe disease."}},"tag":"EXP"},{"id":479,"details":{"paperId":"efa9cd95d23a5a73a060a1379740e6fe6de91753","externalIds":{"MAG":"2789414316","PubMedCentral":"5825926","DOI":"10.3389/fimmu.2018.00226","CorpusId":"3444991","PubMed":"29515570"},"title":"Factors Affecting the Immunity to Respiratory Syncytial Virus: From Epigenetics to Microbiome","abstract":"Respiratory syncytial virus (RSV) is a common pathogen that infects virtually all children by 2 years of age and is the leading cause of hospitalization of infants worldwide. While most children experience mild symptoms, some children progress to severe lower respiratory tract infection. Those children with severe disease have a much higher risk of developing childhood wheezing later in life. Many risk factors are known to result in exacerbated disease, including premature birth and early age of RSV infection, when the immune system is relatively immature. The development of the immune system before and after birth may be altered by several extrinsic and intrinsic factors that could lead to severe disease predisposition in children who do not exhibit any currently known risk factors. Recently, the role of the microbiome and the resulting metabolite profile has been an area of intense study in the development of lung disease, including viral infection and asthma. This review explores both known risk factors that can lead to severe RSV-induced disease as well as emerging topics in the development of immunity to RSV and the long-term consequences of severe infection.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This review explores both known risk factors that can lead to severe RSV-induced disease as well as emerging topics in the development of immunity to RSV and the long-term consequences of severe infection."}},"tag":"EXP"},{"id":92,"details":{"paperId":"f10065c38f6937735160b0f26bfaee34cc6f1781","externalIds":{"MAG":"1995994984","DOI":"10.1016/j.virol.2014.05.032","CorpusId":"5210455","PubMed":"25010481"},"title":"Factors affecting de novo RNA synthesis and back-priming by the respiratory syncytial virus polymerase.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Findings indicate that during infection, the RdRp is governed between de novo RNA synthesis and back-priming by RNA sequence and environment, including a factor missing from the in vitro assay."}},"tag":"EXP"},{"id":111,"details":{"paperId":"2778b9d85904dec22b9121205aea55f12ed69db7","externalIds":{"MAG":"1562992265","DOI":"10.1016/S0021-9258(18)81623-4","CorpusId":"21865711","PubMed":"2732224"},"title":"Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that the fatty acid is covalently attached to the F1 subunit of the fusion glycoprotein of respiratory syncytial virus and the identification of the attachment site is the covalent attachment site for palmitate."}},"tag":"EXP"},{"id":323,"details":{"paperId":"3ae62509be04a81b04de863ab12149dbf0c081e3","externalIds":{"MAG":"2059648382","DOI":"10.1128/JVI.03619-14","CorpusId":"32847110","PubMed":"25653447"},"title":"Fine Mapping and Characterization of the L-Polymerase-Binding Domain of the Respiratory Syncytial Virus Phosphoprotein","abstract":"ABSTRACT The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and the formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The P domain is mapped and characterized and allowed to define a new target for the development of drugs against RSV, extending the understanding of the mechanism of action of RSV RNA polymerase and allowing for the formation of an active L-P complex."}},"tag":"EXP"},{"id":129,"details":{"paperId":"9aa76df0cc0bfe788d2168e00424c5132b32d430","externalIds":{"MAG":"2076341407","DOI":"10.1021/bi401029q","CorpusId":"206847698","PubMed":"23984912"},"title":"Fine modulation of the respiratory syncytial virus M2-1 protein quaternary structure by reversible zinc removal from its Cys(3)-His(1) motif.","abstract":"Human respiratory syncytial virus (hRSV) is a worldwide distributed pathogen that causes respiratory disease mostly in infants and the elderly. The M2-1 protein of hRSV functions as a transcription antiterminator and partakes in virus particle budding. It is present only in Pneumovirinae, namely, Pneumovirus (RSV) and Metapneumovirus, making it an interesting target for specific antivirals. hRSV M2-1 is a tight tetramer bearing a Cys3-His1 zinc-binding motif, present in Ebola VP30 protein and some eukaryotic proteins, whose integrity was shown to be essential for protein function but without a biochemical mechanistic basis. We showed that removal of the zinc atom causes dissociation to a monomeric apo-M2-1 species. Surprisingly, the secondary structure and stability of the apo-monomer is indistinguishable from that of the M2-1 tetramer. Dissociation reported by a highly sensitive tryptophan residue is much increased at pH 5.0 compared to pH 7.0, suggesting a histidine protonation cooperating in zinc removal. The monomeric apo form binds RNA at least as well as the tetramer, and this interaction is outcompeted by the phosphoprotein P, the RNA polymerase cofactor. The role of zinc goes beyond stabilization of local structure, finely tuning dissociation to a fully folded and binding competent monomer. Removal of zinc is equivalent to the disruption of the motif by mutation, only that the former is potentially reversible in the cellular context. Thus, this process could be triggered by a natural chelator such as glutathione or thioneins, where reversibility strongly suggests a modulatory role in the participation of M2-1 in the assembly of the polymerase complex or in virion budding.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that removal of the zinc atom causes dissociation to a monomeric apo-M2-1 species, and this process could be triggered by a natural chelator such as glutathione or thioneins, where reversibility strongly suggests a modulatory role in the participation of M1-1 in the assembly of the polymerase complex or in virion budding."}},"tag":"EXP"},{"id":286,"details":{"paperId":"a50aab3a90de4f022ae735c9c97db3aa5ac0e69f","externalIds":{"MAG":"2800199373","DOI":"10.1128/JVI.00621-18","CorpusId":"13684566","PubMed":"29743373"},"title":"Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity","abstract":"RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development. ABSTRACT The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The essential residues identified in the apical domain of F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development."}},"tag":"EXP"},{"id":338,"details":{"paperId":"92a880f03e3241bd53a0ecba13f7ea2e30551994","externalIds":{"MAG":"2321395094","DOI":"10.1128/jvi.67.2.1090-1093.1993","CorpusId":"10725950","PubMed":"7678299"},"title":"Frequent polymerase errors observed in a restricted area of clones derived from the attachment (G) protein gene of respiratory syncytial virus","abstract":"Sequence analysis of a large number of clones derived from the carboxy-terminal one-third of the attachment (G) protein gene of subgroup A respiratory syncytial viruses revealed a region very prone to polymerase errors which resulted mainly in frameshifts because of the insertion or deletion of adenosine residues in some but not all runs of such residues. Such mutations were detected in 14% of clones derived from mRNA, 58% of clones derived from genomic-sense RNA, and 50% of clones derived from in vitro-transcribed RNA. This phenomenon appears to be dependent on the template sequence.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Sequence analysis of a large number of clones derived from the carboxy-terminal one-third of the attachment (G) protein gene of subgroup A respiratory syncytial viruses revealed a region very prone to polymerase errors which resulted mainly in frameshifts because of the insertion or deletion of adenosine residues in some but not all runs of such residues."}},"tag":"EXP"},{"id":319,"details":{"paperId":"a8f83072191d22c5af71b4802e0d29c0300e4738","externalIds":{"MAG":"2022993968","DOI":"10.1128/JVI.02717-06","CorpusId":"10498435","PubMed":"17494063"},"title":"Function of the Respiratory Syncytial Virus Small Hydrophobic Protein","abstract":"ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, encodes a small hydrophobic (SH) protein of unknown function. Parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, also encodes an SH protein, which inhibits tumor necrosis factor alpha (TNF-α) signaling. In this study, recombinant PIV5 viruses without their own SH but containing RSV SH (from RSV strain A2 or B1) in its place (PIV5ΔSH-RSV SH) and RSV lacking its own SH (RSVΔSH) were generated and analyzed. The results indicate that the SH protein of RSV has a function similar to that of PIV5 SH and that it can inhibit TNF-α signaling.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results indicate that the SH protein of RSV has a function similar to that of PIV5 SH and that it can inhibit TNF-α signaling."}},"tag":"EXP"},{"id":291,"details":{"paperId":"20822fddb02440fb0db1775271540bb972432a2e","externalIds":{"MAG":"1598758802","DOI":"10.1128/JVI.01045-15","CorpusId":"23039992","PubMed":"26018171"},"title":"Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein","abstract":"ABSTRACT There are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup, isolates are further divided into clades. Several years ago, multiple subgroup B isolates which contained a duplication of 60 nucleotides in the glycoprotein (G) gene were described. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene or a consensus BA G gene lacking the duplication (GΔdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness. IMPORTANCE Respiratory syncytial virus (RSV) is an important pathogen for infants for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is determined that the duplicated region in the BA strain G protein functions during virus attachment to cells and that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication."}},"tag":"EXP"},{"id":364,"details":{"paperId":"c57fad0e26dae275e3ca61251ff5f642ddd7a2fc","externalIds":{"MAG":"2003233817","DOI":"10.1128/jvi.74.13.6006-6014.2000","CorpusId":"20558637","PubMed":"10846082"},"title":"Functional Analysis of the Genomic and Antigenomic Promoters of Human Respiratory Syncytial Virus","abstract":"ABSTRACT The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3′ ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3′ genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3′-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3′ end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancingcis-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3′ end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3′ and 5′ ends. The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3′ end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription."}},"tag":"EXP"},{"id":502,"details":{"paperId":"560531f10cf4d05dfbe4e47ebbcc92801535e3ab","externalIds":{"PubMedCentral":"8310105","DOI":"10.3390/v13071214","CorpusId":"236319986","PubMed":"34372490"},"title":"Functional Features of the Respiratory Syncytial Virus G Protein","abstract":"Respiratory syncytial virus (RSV) is a major cause of serious lower respiratory tract infections in children < 5 years of age worldwide and repeated infections throughout life leading to serious disease in the elderly and persons with compromised immune, cardiac, and pulmonary systems. The disease burden has made it a high priority for vaccine and antiviral drug development but without success except for immune prophylaxis for certain young infants. Two RSV proteins are associated with protection, F and G, and F is most often pursued for vaccine and antiviral drug development. Several features of the G protein suggest it could also be an important to vaccine or antiviral drug target design. We review features of G that effect biology of infection, the host immune response, and disease associated with infection. Though it is not clear how to fit these together into an integrated picture, it is clear that G mediates cell surface binding and facilitates cellular infection, modulates host responses that affect both immunity and disease, and its CX3C aa motif contributes to many of these effects. These features of G and the ability to block the effects with antibody, suggest G has substantial potential in vaccine and antiviral drug design.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV proteins G mediates cell surface binding and facilitates cellular infection, modulates host responses that affect both immunity and disease, and its CX3C aa motif contributes to many of these effects."}},"tag":"EXP"},{"id":27,"details":{"paperId":"44d4c2828bfa1e130dc7029b5ee03497e0992b83","externalIds":{"MAG":"2419233968","DOI":"10.1007/978-1-59745-393-6_5","CorpusId":"34966165","PubMed":"17502671"},"title":"Functional analysis of the N-linked glycans within the fusion protein of respiratory syncytial virus.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The respiratory syncytial virus fusion protein is initially expressed as a single polypeptide chain but subsequently undergoes posttranslational cleavage-by-cell protease activity to produce the F1 and F2 subunits, which are modified by the addition of N-linked glycans."}},"tag":"EXP"},{"id":340,"details":{"paperId":"1fb4c8d3967f618470468816e3c922a7ad9f8850","externalIds":{"MAG":"2110993167","DOI":"10.1128/jvi.69.4.2412-2419.1995","CorpusId":"36782060","PubMed":"7884888"},"title":"Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication","abstract":"The RNA-dependent RNA polymerase of human respiratory syncytial (RS) virus was expressed in a functional form from a cDNA clone. Coexpression of the viral polymerase (L) protein, phosphoprotein (P), and nucleocapsid (N) protein allowed us to develop a system for expression and recovery of replicable RS virus RNA entirely from cDNA clones. cDNA clones of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptides. Two types of RS virus genomic RNA analogs were expressed from an intracellular transcription plasmid that directed the synthesis of RNAs with defined 5' and 3' ends. One analog included the authentic 5' and 3' termini of the genome, and the second contained the authentic 5' terminus and its complement at the 3' terminus as found in copyback defective interfering RNAs of other negative-strand RNA viruses. Both types of genomic analogs were encapsidated and replicated in cells expressing the RS virus N, P, and L proteins. Omission of any of the three viral proteins abrogated replication, thereby defining the N, P, and L proteins as the minimal trans-acting proteins required for RNA replication. This system has the advantages that expression occurs at a level sufficient to allow direct biochemical analysis of the products of RNA replication and that neither the use of reporter genes nor wild-type RS helper virus is required. These features allow analysis of both cis- and trans-acting factors involved in the control of replication of RS virus RNA.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A system for expression and recovery of replicable RS virus RNA entirely from cDNA clones is developed that has the advantages that expression occurs at a level sufficient to allow direct biochemical analysis of the products of RNA replication and that neither the use of reporter genes nor wild-type RS helper virus is required."}},"tag":"EXP"},{"id":228,"details":{"paperId":"f2fdd6d054dd65e98756e370f5dd614680c22aa3","externalIds":{"MAG":"2129850004","DOI":"10.1099/0022-1317-82-6-1375","CorpusId":"25232866","PubMed":"11369882"},"title":"Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells.","abstract":"The intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0(EHs)) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0(EHr)). The size and glycosylation state of F0(EHr) were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0(EHr) was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0(EHr) was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that activation of the F protein is required for the efficient formation of RSV filaments."}},"tag":"EXP"},{"id":333,"details":{"paperId":"ba211e50cbdf77da667aab852025868cf1e8b9f1","externalIds":{"MAG":"1550534458","DOI":"10.1128/jvi.62.7.2228-2233.1988","CorpusId":"222251470"},"title":"Further characterization of the soluble form of the G glycoprotein of respiratory syncytial virus","abstract":"A soluble form of the G glycoprotein, the attachment protein, of respiratory syncytial virus is shed from infected HEp-2 cells. The Gs proteins of the Long and 18537 strains have apparent molecular sizes of 82 and 71 kilodaltons, respectively, 6 to 9 kilodaltons smaller than the virion-associated forms (Gv). The Gs protein of the Long strain was further characterized. Approximately one in six of all of the radiolabeled G molecules in these cultures at 24 h postinfection was present as the Gs protein. The Gs protein was clearly evident in culture fluids at 6 h postinfection, but the Gv protein could not be discerned until 12 h after infection, an observation that is consistent with the 12-h eclipse period for respiratory syncytial virus. Therefore, the Gs protein is shed, in part at least, from intact, infected cells and before the appearance of progeny virus. The appearance of a smaller Gs protein (74 kilodaltons) in fluids of infected calls which were incubated with tunicamycin shows that addition of N-linked oligosaccharides is not required for the genesis and shedding of the Gs protein. Sequencing of the amino terminus of purified Gs protein revealed two different termini, whose generations are consistent with cleavages of the full-length G protein between amino acids 65 and 66 and between residues 74 and 75. This result suggests that the Gs protein is present in two different forms which lack the proposed intracytoplasmic and transmembrane domains of the full-length G protein.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The appearance of a smaller Gs protein (74 kilodaltons) in fluids of infected calls which were incubated with tunicamycin shows that addition of N-linked oligosaccharides is not required for the genesis and shedding of the Gsprotein."}},"tag":"EXP"},{"id":430,"details":{"paperId":"16ad2bf53fd54d62aa2993ef1ec3665b1e1b8819","externalIds":{"MAG":"2249695594","DOI":"10.1242/jcs.032169","CorpusId":"16990201","PubMed":"18946025"},"title":"Fusion-pore expansion during syncytium formation is restricted by an actin network","abstract":"Cell-cell fusion in animal development and in pathophysiology involves expansion of nascent fusion pores formed by protein fusogens to yield an open lumen of cell-size diameter. Here we explored the enlargement of micron-scale pores in syncytium formation, which was initiated by a well-characterized fusogen baculovirus gp64. Radial expansion of a single or, more often, of multiple fusion pores proceeds without loss of membrane material in the tight contact zone. Pore growth requires cell metabolism and is accompanied by a local disassembly of the actin cortex under the pores. Effects of actin-modifying agents indicate that the actin cortex slows down pore expansion. We propose that the growth of the strongly bent fusion-pore rim is restricted by a dynamic resistance of the actin network and driven by membrane-bending proteins that are involved in the generation of highly curved intracellular membrane compartments.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is proposed that the growth of the strongly bent fusion-pore rim is restricted by a dynamic resistance of the actin network and driven by membrane-bending proteins that are involved in the generation of highly curved intracellular membrane compartments."}},"tag":"EXP"},{"id":300,"details":{"paperId":"fc72c21bda84a09cd08421ef2d371ef04f3b20f2","externalIds":{"MAG":"3048527268","DOI":"10.1128/JVI.01407-20","CorpusId":"222348243","PubMed":"32796072"},"title":"GBP5 Is an Interferon-Induced Inhibitor of Respiratory Syncytial Virus","abstract":"RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection are necessary to develop an effective strategy to control RSV infection. Here, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host. ABSTRACT Guanylate binding protein 5 (GBP5) belongs to the GTPase subfamily, which is mainly induced by interferon gamma (IFN-γ) and is involved in many important cellular processes, including inflammasome activation and innate immunity against a wide variety of microbial pathogens. However, it is unknown whether GBP5 inhibits respiratory syncytial virus (RSV) infection. In this study, we identified GBP5 as an effector of the anti-RSV activity of IFN-γ and found that in children, the weaker immune response, especially the weaker IFN-γ response and the decreased GBP5 expression, leads to RSV susceptibility. Furthermore, we revealed that GBP5 reduced the cell-associated levels of the RSV small hydrophobic (SH) protein, which was identified as a viroporin. In contrast, overexpression of the SH protein rescued RSV replication in the presence of GBP5. The GBP5-induced decrease in intracellular SH protein levels is because GBP5 promotes the release of the SH protein into the cell culture. Moreover, the GBP5 C583A mutants with changes at the C terminus or the GBP5 ΔC mutant lacking the C-terminal region, which impairs GBP5 localization in the Golgi, could not inhibit RSV infection, whereas the GTPase-defective GBP5 maintained RSV inhibition, suggesting that Golgi localization but not the GTPase activity of GBP5 is required for RSV inhibition. Interestingly, we found that RSV infection or RSV G protein downregulates GBP5 expression by upregulating DZIP3, an E3 ligase, which induces GBP5 degradation through the K48 ubiquitination and proteasomal pathways. Thus, this study reveals a complicated interplay between host restrictive factor GBP5 and RSV infection and provides important information for understanding the pathogenesis of RSV. IMPORTANCE RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection are necessary to develop an effective strategy to control RSV infection. Here, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"GBP5 is identified as an effector of the anti-RSV activity of IFN-γ and found that in children, the weaker immune response, especially the weaker IFn-γ response and the decreased GBP5 expression, leads to RSV susceptibility."}},"tag":"EXP"},{"id":367,"details":{"paperId":"c717f681e568b69a19815557186256ba0a566ec2","externalIds":{"MAG":"2102146833","DOI":"10.1128/JVI.74.22.10508-10513.2000","CorpusId":"20115578","PubMed":"11044095"},"title":"Glycosaminoglycan Sulfation Requirements for Respiratory Syncytial Virus Infection","abstract":"ABSTRACT Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant green fluorescent protein-expressing RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in N-sulfation was three times less efficient than infection of the parental CHO cell line, indicating thatN-sulfation is important. In contrast, infection of a cell line deficient in 2-O-sulfation was as efficient as infection of the parental cell line, indicating that 2-O-sulfation is not required for RSV infection. Incubating RSV with the purified soluble heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified heparin chains that lack theirN-, C6-O-, or C2-O-sulfate groups. Only heparin chains lacking the N-sulfate group lost the ability to neutralize infection, confirming thatN-sulfation, but not C6-O- or C2-O-sulfation, is important for RSV infection. Analysis of heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required. This specificity indicates that the role of cell surface GAGs in RSV infection is not based on a simple charge interaction between the virus and sulfate groups but instead involves a specific GAG structural configuration that includes N-sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264–275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required."}},"tag":"EXP"},{"id":467,"details":{"paperId":"9b111e8063f00cdc91edf9b5d49fb1683ff23537","externalIds":{"PubMedCentral":"8804925","DOI":"10.15252/embj.2021109728","CorpusId":"236938843","PubMed":"34935163"},"title":"Helical ordering of envelope‐associated proteins and glycoproteins in respiratory syncytial virus","abstract":"Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Treatments for RSV disease are however limited and efforts to produce an effective vaccine have so far been unsuccessful. Understanding RSV virion structure is an important prerequisite for developing interventions to treat or prevent infection but has been challenging because of the fragility of virions propagated in cell culture. Here we show, using cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) of RSV particles cultivated directly on transmission electron microscopy (TEM) grids, that there is extensive helical symmetry in RSV filamentous virions. We have calculated a 16 Å resolution three-dimensional reconstruction of the viral envelope, targeting the matrix protein (M) that forms an endoskeleton below the viral membrane. These data define a helical lattice of M proteins, showing how M is oriented relative to the viral envelope and that helical ordering of viral glycoproteins that stud the viral envelope is coordinated by the M layer. Moreover, the helically ordered viral glycoproteins in RSV filamentous virions cluster in pairs, which may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV filamentous virions. Overall, the structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Structural data obtained provides molecular insight into the organization of the virion and the mechanism of its assembly and may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development."}},"tag":"EXP"},{"id":353,"details":{"paperId":"c991ffc6d4fe28b3381588be1b7ec99107b662a0","externalIds":{"MAG":"2111143976","DOI":"10.1128/JVI.72.9.7221-7227.1998","CorpusId":"30125107","PubMed":"9696816"},"title":"Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity In Vitro","abstract":"ABSTRACT Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results prove that heparin-like structures identified on RSV play a major role in early stages of infection, and that the RSV G protein is the attachment protein, and two anti-heparan sulfate antibodies specifically bound to this protein."}},"tag":"EXP"},{"id":35,"details":{"paperId":"399160d326a3e6be2462b9a49ad4334d00c88d67","externalIds":{"MAG":"1999756472","DOI":"10.1007/s007050050156","CorpusId":"19048460","PubMed":"9229012"},"title":"Heparin-dependent attachment ofrespiratory syncytial virus (RSV) to host cells","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that heparin (ED50 = 0.32 ±), but not heparan sulphate or chondroitin sulphate C is able to inhibit in vitro infection of cells by respiratory syncytial virus (RSV), and heparIn-like proteoglycans showed properties required for attachment of RSV to host cells."}},"tag":"EXP"},{"id":124,"details":{"paperId":"427626e13fc3f7ab6788128d41b2e293380f677e","externalIds":{"MAG":"2055974092","DOI":"10.1021/BI000471Y","CorpusId":"10041777","PubMed":"10995236"},"title":"Heptad-repeat regions of respiratory syncytial virus F1 protein form a six-membered coiled-coil complex.","abstract":"The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques. Two heptad-repeat (HR) regions within F(1) were shown to interact. Proteinase-K digestion experiments highlight the HR1 region (located proximal to the fusion peptide sequence) of the F(1) protein to which an HR2-derived (located proximal to the membrane-spanning domain) peptide binds, thus protecting both the protein and peptide from digestion. Solution-phase analysis of HR1-derived peptides shows that these peptides adopt helical secondary structure as measured by circular dichroism. Sedimentation equilibrium studies indicate that these HR1 peptides self-associate in a monomer/trimer equilibrium with an association constant of 5.2 x 10(8) M(-2). In contrast, HR2-derived peptides form random monomers in solution. CD analysis of mixtures containing peptides from the two regions demonstrate their propensity to interact and form a very stable (T(m) = 87 degrees C), helical (86% helicity) complex comprised of three HR1 and three HR2 members.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques and two heptad-repeat (HR) regions within F( 1) were shown to interact."}},"tag":"EXP"},{"id":483,"details":{"paperId":"7bd17be9a42c93dc2df43162e410b83faf6ea287","externalIds":{"MAG":"2970924044","PubMedCentral":"6753334","DOI":"10.3389/fimmu.2019.02152","CorpusId":"202550211","PubMed":"31572372"},"title":"Host Components Contributing to Respiratory Syncytial Virus Pathogenesis","abstract":"Respiratory syncytial virus (RSV) is the most prevalent viral etiological agent of acute respiratory tract infection. Although RSV affects people of all ages, the disease is more severe in infants and causes significant morbidity and hospitalization in young children and in the elderly. Host factors, including an immature immune system in infants, low lymphocyte levels in patients under 5 years old, and low levels of RSV-specific neutralizing antibodies in the blood of adults over 65 years of age, can explain the high susceptibility to RSV infection in these populations. Other host factors that correlate with severe RSV disease include high concentrations of proinflammatory cytokines such as interleukins (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and thymic stromal lymphopoitein (TSLP), which are produced in the respiratory tract of RSV-infected individuals, accompanied by a strong neutrophil response. In addition, data from studies of RSV infections in humans and in animal models revealed that this virus suppresses adaptive immune responses that could eliminate it from the respiratory tract. Here, we examine host factors that contribute to RSV pathogenesis based on an exhaustive review of in vitro infection in humans and in animal models to provide insights into the design of vaccines and therapeutic tools that could prevent diseases caused by RSV.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Examination of host factors that contribute to RSV pathogenesis based on an exhaustive review of in vitro infection in humans and in animal models is examined to provide insights into the design of vaccines and therapeutic tools that could prevent diseases caused by RSV."}},"tag":"EXP"},{"id":400,"details":{"paperId":"5cac24f82965788272dbd4af43b311868afd5677","externalIds":{"MAG":"3090115399","PubMedCentral":"7527724","DOI":"10.1128/mBio.01869-20","CorpusId":"222168616","PubMed":"32994321"},"title":"Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production","abstract":"The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process. ABSTRACT Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nongly cosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment."}},"tag":"EXP"},{"id":12,"details":{"paperId":"49bde960dcc21cb4012a8f3c130621743f4359fe","externalIds":{"MAG":"1987769422","DOI":"10.1006/VIRO.1996.0379","CorpusId":"6321280","PubMed":"8661440"},"title":"Host cell effect upon glycosylation and antigenicity of human respiratory syncytial virus G glycoprotein.","abstract":"Infection of different human epithelial cell lines with human respiratory syncytial virus (HRSV) revealed significant differences in the electrophoretic mobility of the viral attachment glycoprotein (G). Cell-type specific differences in G protein glycosylation were observed with certain lectins and sugar-specific reagents. Furthermore, substantial changes in the reactivity of the G glycoprotein with anti-G monoclonal antibodies were associated to the infected cell type. Strain-specific epitopes--present in a limited number of HRSV isolates of the same antigenic group--were particularly susceptible to cell-type-specific modifications of the mature G protein. Some of these epitopes, which were either exposed in the unglycosylated precursor or reproduced with synthetic peptides, were nonetheless masked in the mature G protein expressed in certain cell lines. Antigenic and electrophoretic mobility changes of the G glycoprotein were reverted in extracts of HEp-2 cells infected with HRSV grown in other cell types, indicating that phenotypic traits rather than selection of variants were associated to the above stated changes. These results highlight the importance of cell-type-specific modifications for HSRV G glycoprotein antigenicity and raise questions about the actual antigenic structure of this molecule when HRSV replicates in the respiratory tract.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results highlight the importance of cell-type-specific modifications for HSRV G glycoprotein antigenicity and raise questions about the actual antigenic structure of this molecule when HRSV replicates in the respiratory tract."}},"tag":"EXP"},{"id":503,"details":{"paperId":"f77f55f19dbc4252933ffbe2df6b18486159d27c","externalIds":{"PubMedCentral":"8874859","DOI":"10.3390/v14020419","CorpusId":"246984412","PubMed":"35216012"},"title":"How RSV Proteins Join Forces to Overcome the Host Innate Immune Response","abstract":"Respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Although several pattern recognition receptors (PRRs) can sense RSV-derived pathogen-associated molecular patterns (PAMPs), infection with RSV is typically associated with low to undetectable levels of type I interferons (IFNs). Multiple RSV proteins can hinder the host’s innate immune response. The main players are NS1 and NS2 which suppress type I IFN production and signalling in multiple ways. The recruitment of innate immune cells and the production of several cytokines are reduced by RSV G. Next, RSV N can sequester immunostimulatory proteins to inclusion bodies (IBs). N might also facilitate the assembly of a multiprotein complex that is responsible for the negative regulation of innate immune pathways. Furthermore, RSV M modulates the host’s innate immune response. The nuclear accumulation of RSV M has been linked to an impaired host gene transcription, in particular for nuclear-encoded mitochondrial proteins. In addition, RSV M might also directly target mitochondrial proteins which results in a reduced mitochondrion-mediated innate immune recognition of RSV. Lastly, RSV SH might prolong the viral replication in infected cells and influence cytokine production.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide and the main players are NS1 and NS2 which suppress type I IFN production and signalling in multiple ways."}},"tag":"EXP"},{"id":498,"details":{"paperId":"45d3b6128146d7df335e96fb8257644f0a5fca14","externalIds":{"PubMedCentral":"6316657","MAG":"2905476218","DOI":"10.3390/v10120716","CorpusId":"56178152","PubMed":"30558248"},"title":"Human Respiratory Syncytial Virus NS 1 Targets TRIM25 to Suppress RIG-I Ubiquitination and Subsequent RIG-I-Mediated Antiviral Signaling","abstract":"Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling."}},"tag":"EXP"},{"id":402,"details":{"paperId":"d531df49b77873bfee59c3ad087b0c0fdf9b0255","externalIds":{"PubMedCentral":"8764521","DOI":"10.1128/mbio.03528-21","CorpusId":"246001195","PubMed":"35038909"},"title":"Human Respiratory Syncytial Virus NS2 Protein Induces Autophagy by Modulating Beclin1 Protein Stabilization and ISGylation","abstract":"Understanding host-virus interactions is essential for the development of effective interventions against respiratory syncytial virus (RSV), a paramyxovirus that is a leading cause of viral pneumonia in infants. RSV induces autophagy following infection, although the viral factors involved in this mechanism are unknown. ABSTRACT Paramyxoviruses such as respiratory syncytial virus (RSV) are the leading cause of pneumonia in infants, the elderly, and immunocompromised individuals. Understanding host-virus interactions is essential for the development of effective interventions. RSV induces autophagy to modulate the immune response. The viral factors and mechanisms underlying RSV-induced autophagy are unknown. Here, we identify the RSV nonstructural protein NS2 as the virus component mediating RSV-induced autophagy. We show that NS2 interacts and stabilizes the proautophagy mediator Beclin1 by preventing its degradation by the proteasome. NS2 further impairs interferon-stimulated gene 15 (ISG15)-mediated Beclin1 ISGylation and generates a pool of “hypo-ISGylated” active Beclin1 to engage in functional autophagy. Studies with NS2-deficient RSV revealed that NS2 contributes to RSV-mediated autophagy during infection. The present study is the first report to show direct activation of autophagy by a paramyxovirus nonstructural protein. We also report a new viral mechanism for autophagy induction wherein the viral protein NS2 promotes hypo-ISGylation of Beclin1 to ensure availability of active Beclin1 to engage in the autophagy process. IMPORTANCE Understanding host-virus interactions is essential for the development of effective interventions against respiratory syncytial virus (RSV), a paramyxovirus that is a leading cause of viral pneumonia in infants. RSV induces autophagy following infection, although the viral factors involved in this mechanism are unknown. Here, we identify the RSV nonstructural protein 2 (NS2) as the virus component involved in autophagy induction. NS2 promotes autophagy by interaction with and stabilization of the proautophagy mediator Beclin1 and by impairing its ISGylation to overcome autophagy inhibition. To the best of our knowledge, this is the first report of a viral protein regulating the autophagy pathway by modulating ISGylation of autophagy mediators. Our studies highlight a direct role of a paramyxovirus nonstructural protein in activating autophagy by interacting with the autophagy mediator Beclin1. NS2-mediated regulation of the autophagy and ISGylation processes is a novel function of viral nonstructural proteins to control the host response against RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This study identifies RSV nonstructural protein 2 (NS2) as the virus component mediating RSV-induced autophagy induction and shows the first report of a viral protein regulating the autophile pathway by modulating ISGylation of autophile mediators."}},"tag":"EXP"},{"id":316,"details":{"paperId":"1ea77c4e2a84aa57e20aa4a385acdb7dc67e7475","externalIds":{"MAG":"2114884151","DOI":"10.1128/JVI.02434-08","CorpusId":"22918230","PubMed":"19193793"},"title":"Human Respiratory Syncytial Virus Nonstructural Protein NS2 Antagonizes the Activation of Beta Interferon Transcription by Interacting with RIG-I","abstract":"ABSTRACT A wide variety of RNA viruses have been shown to produce proteins that inhibit interferon (IFN) production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to block IFN signaling by causing the proteasomal degradation of STAT2. In addition, recombinant RSVs lacking either NS1 or NS2 induce more IFN production than wild-type (wt) RSV in infected cells. However, the mechanisms by which the NS proteins perform this function are unknown. In this study, we focused on defining the mechanism by which NS2 inhibits the induction of IFN transcription. We find that NS2 is required for the early inhibition of IFN transcription since the infection of cells with NS2-deletion RSV resulted in a higher level of IRF3 activation at early time points postinfection compared with that of wt or NS1-deletion RSV infection. In addition, NS2 expression inhibits IFN transcription induced by both the RIG-I and TLR3 pathways. Furthermore, we show that NS2 inhibits RIG-I-mediated IFN promoter activation by binding to the N-terminal CARD of RIG-I and inhibiting its interaction with the downstream component MAVS (IPS-1, VISA, Cardif). Thus, the RSV NS2 protein is a multifunctional IFN antagonist that targets specific components of both the IFN induction and IFN signaling pathways.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The RSV NS2 protein is a multifunctional IFN antagonist that targets specific components of both the IFN induction and IFN signaling pathways."}},"tag":"EXP"},{"id":274,"details":{"paperId":"e49abf873d3e57c20b2dca88f6bf613182f3ec77","externalIds":{"MAG":"2050378052","DOI":"10.1128/JVI.00215-12","CorpusId":"7335998","PubMed":"22623778"},"title":"Human Respiratory Syncytial Virus Nucleoprotein and Inclusion Bodies Antagonize the Innate Immune Response Mediated by MDA5 and MAVS","abstract":"ABSTRACT Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response."}},"tag":"EXP"},{"id":152,"details":{"paperId":"dfcaa02c9e7553c1b1330925c18fabf2ccad5d79","externalIds":{"MAG":"2478760109","PubMedCentral":"7171722","DOI":"10.1055/s-0036-1584799","CorpusId":"3634817","PubMed":"27486734"},"title":"Human Respiratory Syncytial Virus: Infection and Pathology","abstract":"Abstract The human respiratory syncytial virus (hRSV) is by far the major cause of acute lower respiratory tract infections (ALRTIs) worldwide in infants and children younger than 2 years. The overwhelming number of hospitalizations due to hRSV-induced ALRTI each year is due, at least in part, to the lack of licensed vaccines against this virus. Thus, hRSV infection is considered a major public health problem and economic burden in most countries. The lung pathology developed in hRSV-infected individuals is characterized by an exacerbated proinflammatory and unbalanced Th2-type immune response. In addition to the adverse effects in airway tissues, hRSV infection can also cause neurologic manifestations in the host, such as seizures and encephalopathy. Although the origins of these extrapulmonary symptoms remain unclear, studies with patients suffering from neurological alterations suggest an involvement of the inflammatory response against hRSV. Furthermore, hRSV has evolved numerous mechanisms to modulate and evade the immune response in the host. Several studies have focused on elucidating the interactions between hRSV virulence factors and the host immune system, to rationally design new vaccines and therapies against this virus. Here, we discuss about the infection, pathology, and immune response triggered by hRSV in the host.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The infection, pathology, and immune response triggered by hRSV in the host are discussed, to rationally design new vaccines and therapies against this virus."}},"tag":"OTHER"},{"id":185,"details":{"paperId":"a7003c6a0e75022a3d9121f6859c1a3d5023db09","externalIds":{"MAG":"2074807977","DOI":"10.1080/01652176.1991.9694284","CorpusId":"6998424","PubMed":"2021054"},"title":"Human and bovine respiratory syncytial virus: immunopathologic mechanisms.","abstract":"Human respiratory syncytial virus (HRSV) is the major respiratory tract pathogen of infants and young children. Bovine respiratory syncytial virus (BRSV) is recognised as an important cause of respiratory tract disease in calves. Both of these viruses and their respective diseases share many similarities. Immunopathologic mechanisms have been proposed to be involved in the pathogenesis of respiratory syncytial virus (RSV) infections. This review examines the current understanding of the role of immunopathologic mechanisms in RSV infections. The role of vaccines in inducing hypersensitivity is also examined. Additionally, non-immunopathogenic mechanisms involved in RSV infections are discussed.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The role of vaccines in inducing hypersensitivity is examined and non-immunopathogenic mechanisms involved in RSV infections are discussed."}},"tag":"OTHER"},{"id":428,"details":{"paperId":"761f5d9404210072ed7a930fcd262f9f4de8e47a","externalIds":{"DOI":"10.1186/s13104-015-1836-y","CorpusId":"256005374"},"title":"Human cathelicidin, LL-37, inhibits respiratory syncytial virus infection in polarized airway epithelial cells","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Differences in the efficacy of LL-37 at reducing RSV infection under prophylactic and therapeutic conditions may in part be ascribed to differences in the method of peptide exposure."}},"tag":"EXP"},{"id":102,"details":{"paperId":"3bc645f10899488075aacdce6c22921c47d5bb2d","externalIds":{"MAG":"2066739594","DOI":"10.1016/j.virusres.2013.07.010","CorpusId":"205658546","PubMed":"23892143"},"title":"Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors, and their potential contribution for a HRSV replication model is discussed."}},"tag":"EXP"},{"id":238,"details":{"paperId":"41d365854036322483d44caad320c6401b2ca9aa","externalIds":{"MAG":"2134446020","DOI":"10.1099/VIR.0.19707-0","CorpusId":"25821352","PubMed":"14993657"},"title":"Human respiratory syncytial virus matrix protein is an RNA-binding protein: binding properties, location and identity of the RNA contact residues.","abstract":"The human respiratory syncytial virus (HRSV) matrix (M) protein is a structural internal membrane protein. Here we have shown that, like its orthomyxovirus and rhabdovirus counterparts, it has RNA-binding capacity, as determined by retardation of (32)P-labelled riboprobes in gel electrophoresis, cross-linking with UV light and Northern-Western assays. Its binding to RNA was neither sequence-specific nor showed a length requirement, although it had cooperative kinetics with a K(d) of 25 nM and probably two different types of RNA-binding sites. After preparative cross-linking of (32)P-labelled riboprobes with purified, renatured HRSV Long strain M protein (256 residues), the residues in contact with RNA were located between amino acids 120 and 170, in the central part of the molecule. Lysine (positions 121, 130, 156 and 157) and arginine (position 170) residues located within this region and conserved among pneumovirus M proteins of different origins were found to be essential for RNA contact. M protein expression did not affect the replication and transcription of HRSV RNA analogues in vivo (except when expressed in large amounts), in contrast to the in vitro transcription inhibition described previously. In addition, M protein was found to aggregate into high-molecular-mass oligomers, both in the presence and absence of its RNA-binding activity. The formation of these structures has been related in other viruses to either viral or host-cell RNA metabolism.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Human respiratory syncytial virus M protein expression did not affect the replication and transcription of HRSV RNA analogues in vivo (except when expressed in large amounts), in contrast to the in vitro transcription inhibition described previously."}},"tag":"EXP"},{"id":231,"details":{"paperId":"a763309b9b1d504abbacc9edf9f8c4c240774ce1","externalIds":{"MAG":"1925673037","DOI":"10.1099/JGV.0.000261","CorpusId":"24805664","PubMed":"26253191"},"title":"Human respiratory syncytial virus non-structural protein NS1 modifies miR-24 expression via transforming growth factor-β.","abstract":"Human respiratory syncytial virus (RSV) is a major health challenge in the young and elderly owing to the lack of a safe and effective vaccine and proven antiviral drugs. Understanding the mechanisms by which viral genes and proteins modulate the host response to infection is critical for identifying novel disease intervention strategies. In this study, the RSV non-structural protein NS1 was shown to suppress miR-24 expression during infection. Lack of NS1 was linked to increased expression of miR-24, whilst NS1 overexpression suppressed miR-24 expression. NS1 was found to induce Kruppel-like factor 6 (KLF6), a transcription factor that positively regulates the transforming growth factor (TGF)-b pathway to induce cell cycle arrest. Silencing of KLF6 led to increased miR-24 expression via downregulation of TGF-β. Treatment with exogenous TGF-β suppressed miR-24 expression and induced KLF6. Confocal microscopy showed co-localization of KLF6 and RSV NS1. These findings indicated that RSV NS1 interacts with KLF6 and modulates miR-24 expression and TGF-β, which facilitates RSV replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV NS1 interacts with KLF6 and modulates miR-24 expression and TGF-β, which facilitates RSV replication, and is found to induce Kruppel-like factor 6 (KLF6), a transcription factor that positively regulates the transforming growth factor-b pathway to induce cell cycle arrest."}},"tag":"EXP"},{"id":36,"details":{"paperId":"4e48957cfc3f0ed59efa743b0eed3287dcf294cc","externalIds":{"MAG":"1968550176","DOI":"10.1007/s007050170009","CorpusId":"21542391","PubMed":"11811686"},"title":"Human respiratory syncytial virus surface glycoproteins F, G and SH form an oligomeric complex","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data suggest that RSV F, G and SH glycoproteins can form an oligomeric complex within infected cells and this complex has a higher affinity for heparin than either G or F protein alone."}},"tag":"EXP"},{"id":404,"details":{"paperId":"3ef0449e7b76d95f4a6dd2521d060d0d940f5ff5","externalIds":{"MAG":"2163881059","DOI":"10.1136/thoraxjnl-2012-202182","CorpusId":"2032120","PubMed":"23229815"},"title":"Human respiratory syncytial virus viroporin SH: a viral recognition pathway used by the host to signal inflammasome activation","abstract":"Background Respiratory syncytial virus (RSV) remains the leading cause of serious viral bronchiolitis and pneumonia in infants and young children throughout the world. The burden of disease is significant, with 70% of all infants being infected with RSV within the first year of their life. 40% of those children discharged from hospital have recurrent, repeated respiratory symptoms and wheezing for at least 10 years. The infection is also an important illness in the elderly and immunocompromised individuals. Ongoing symptoms relate to continued lung inflammation. One cytokine that is associated with RSV infection is IL-1β, but the mechanism of activation remain unclear. Objectives In the current study, we set out to decipher the molecular mechanisms of RSV-induced inflammasome activation. Methods and results Using deletion mutants of the virus and measuring IL-1β secretion, as well as caspase 1 expression via western blotting, we demonstrate that the NLRP3 inflammasome is activated through the small hydrophobic (SH) RSV viroporin which induces membrane permeability to ions or small molecules. Confocal microscopy revealed that during virus infection, SH seems to accumulate within lipid rafts in the Golgi compartments. Conclusions Upon RSV infection, SH gets localised in the cell membranes and intracellular organelle membranes, and then induces permeability by disrupting membrane architecture, thus leading us to believe that formation of viral ion channels in lipid bilayers of cells is a viral recognition pathway used by the host to signal inflammasome activation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Upon RSV infection, SH gets localised in the cell membranes and intracellular organelle membranes, and then induces permeability by disrupting membrane architecture, thus leading us to believe that formation of viral ion channels in lipid bilayers of cells is a viral recognition pathway used by the host to signal inflammasome activation."}},"tag":"EXP"},{"id":143,"details":{"paperId":"9aff9d878e0d3fbba41041d4172b85fe2d92c070","externalIds":{"DOI":"10.1038/s41586-020-2369-7","CorpusId":"256822490"},"title":"IGF1R is an entry receptor for respiratory syncytial virus","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"A mechanism of RSV entry into cells is shown in which outside-in signalling, involving binding of the prefusion RSV-F glycoprotein with the insulin-like growth factor-1 receptor, triggers the activation of protein kinase C zeta (PKCζ)."}},"tag":"EXP"},{"id":324,"details":{"paperId":"7f80a70d4cb3ff2595e38e24cd172ef2dd6efd49","externalIds":{"MAG":"2123144695","DOI":"10.1128/JVI.03666-14","CorpusId":"9318078","PubMed":"25568210"},"title":"Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein","abstract":"ABSTRACT The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae, it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N0-P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N0 monomer. We used this N mutant, designated Nmono, as a substitute for N0 in order to characterize the P regions involved in the N0-P complex formation. Using a series of P fragments, we determined by glutathione S-transferase (GST) pulldown assays that the N and C termini of P are able to interact with Nmono. We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and Nmono in vitro. Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N0-P interaction could constitute a potential antiviral strategy. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N0) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"There is the first evidence, to the authors' knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N, and it is shown that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis."}},"tag":"EXP"},{"id":133,"details":{"paperId":"f456c4554cd65263201728394ce2c614dbfe0bb8","externalIds":{"MAG":"2029410145","DOI":"10.1038/mt.2012.237","CorpusId":"205206730","PubMed":"23183536"},"title":"Identification and functional characterization of tRNA-derived RNA fragments (tRFs) in respiratory syncytial virus infection.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This study presents novel information on the induction of a functional tRF by viral infection by applying deep sequencing to cells infected with human respiratory syncytial virus, the most common cause of bronchiolitis and pneumonia in babies."}},"tag":"EXP"},{"id":384,"details":{"paperId":"733cd5c1943d881eaed0a07fea2283896fa51671","externalIds":{"MAG":"2143904566","DOI":"10.1128/JVI.77.9.5046-5053.2003","CorpusId":"2499582","PubMed":"12692207"},"title":"Identification of Amino Acids That Are Critical to the Processivity Function of Respiratory Syncytial Virus M2-1 Protein","abstract":"ABSTRACT The M2-1 protein of respiratory syncytial virus (RSV) is a transcription processivity factor that is essential for virus replication. The function of RSV M2-1 protein can be examined by using an RSVlacZ minigenome assay in vitro since the expression of the lacZ gene is dependent on M2-1. The M2-1 protein of pneumonia virus of mice (PVM), also a member of the Pneumovirus genus, functions poorly in the RSVlacZ minigenome assay despite conservation of the Cys3-His1 motif at its N terminus and an overall 40% amino acid identity with RSV M2-1. To identify the amino acids responsible for the differences between these two proteins, two chimeric proteins were constructed. The RSV/PVM (RP) M2-1 chimera that contains the N-terminal 30 amino acids from RSV and the remaining C-terminal 148 amino acids from PVM maintained a level of activity at an ca. 36% of RSV M2-1. However, the PVM/RSV (PR) M2-1 chimera with the N-terminal 29 amino acids from PVM and 164 amino acids from RSV had an activity of <5% of RSV M2-1, indicating that the functional determinants are mainly located in the N terminus of M2-1. Mutagenesis of the N terminus of PR M2-1 and RSV M2-1 identified that Leu-16 and Asn-17 of RSV M2-1 are critical to the M2-1 function. In addition, several charged residues in the N terminus of RSV M2-1 also contributed to the functional integrity of M2-1.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The M2-1 protein of pneumonia virus of mice (PVM), also a member of the Pneumovirus genus, functions poorly in the RSVlacZ minigenome assay despite conservation of the Cys3-His1 motif at its N terminus and an overall 40% amino acid identity with RSV M2 -1."}},"tag":"EXP"},{"id":484,"details":{"paperId":"4d8adb137b7fc66e696f0f6b9f99811f2802161a","externalIds":{"PubMedCentral":"9125393","DOI":"10.3389/fmicb.2022.880394","CorpusId":"247146817","PubMed":"35615506"},"title":"Identification of Cytoplasmic Chaperone Networks Relevant for Respiratory Syncytial Virus Replication","abstract":"RNA viruses have limited coding capacity and must therefore successfully subvert cellular processes to facilitate their replication. A fundamental challenge faced by both viruses and their hosts is the ability to achieve the correct folding and assembly of their proteome while avoiding misfolding and aggregation. In cells, this process is facilitated by numerous chaperone systems together with a large number of co-chaperones. In this work, we set out to define the chaperones and co-chaperones involved in the replication of respiratory syncytial virus (RSV). Using an RNAi screen, we identify multiple members of cellular protein folding networks whose knockdown alters RSV replication. The reduced number of chaperones and co-chaperones identified in this work can facilitate the unmasking of specific chaperone subnetworks required for distinct steps of the RSV life cycle and identifies new potential targets for antiviral therapy. Indeed, we show that the pharmacological inhibition of one of the genes identified in the RNAi screen, valosin-containing protein (VCP/p97), can impede the replication of RSV by interfering with the infection cycle at multiple steps.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The reduced number of chaperones and co-chaperones identified in this work can facilitate the unmasking of specific chaperone subnetworks required for distinct steps of the RSV life cycle and identifies new potential targets for antiviral therapy."}},"tag":"EXP"},{"id":393,"details":{"paperId":"514238a8384b64f72d377a9047f699b3622d3d3c","externalIds":{"MAG":"2162049260","DOI":"10.1128/JVI.79.4.2449-2460.2005","CorpusId":"600324","PubMed":"15681446"},"title":"Identification of Internal Sequences in the 3′ Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity","abstract":"ABSTRACT Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3′ end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3′-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di- and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The remainder of the Le region was mapped to identify sequences important for transcription and replication, and the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome."}},"tag":"EXP"},{"id":500,"details":{"paperId":"d8dfa9adf15ef38f62476eac3fd37161e7fe1210","externalIds":{"PubMedCentral":"7915953","DOI":"10.3390/v13020261","CorpusId":"231881865","PubMed":"33567674"},"title":"Identification of RSV Fusion Protein Interaction Domains on the Virus Receptor, Nucleolin","abstract":"Nucleolin is an essential cellular receptor to human respiratory syncytial virus (RSV). Pharmacological targeting of the nucleolin RNA binding domain RBD1,2 can inhibit RSV infections in vitro and in vivo; however, the site(s) on RBD1,2 which interact with RSV are not known. We undertook a series of experiments designed to: document RSV-nucleolin co-localization on the surface of polarized MDCK cells using immunogold electron microscopy, to identify domains on nucleolin that physically interact with RSV using biochemical methods and determine their biological effects on RSV infection in vitro, and to carry out structural analysis toward informing future RSV drug development. Results of immunogold transmission and scanning electron microscopy showed RSV-nucleolin co-localization on the cell surface, as would be expected for a viral receptor. RSV, through its fusion protein (RSV-F), physically interacts with RBD1,2 and these interactions can be competitively inhibited by treatment with Palivizumab or recombinant RBD1,2. Treatment with synthetic peptides derived from two 12-mer domains of RBD1,2 inhibited RSV infection in vitro, with structural analysis suggesting these domains are potentially feasible for targeting in drug development. In conclusion, the identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development."}},"tag":"EXP"},{"id":317,"details":{"paperId":"b9cbfe1b2689a1a05c270712dbaae2bf41f62359","externalIds":{"MAG":"2073045612","DOI":"10.1128/JVI.02472-14","CorpusId":"3197055","PubMed":"25339762"},"title":"Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis","abstract":"ABSTRACT Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein. IMPORTANCE Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity were defined and elucidated a correlation between fusion activity and viral load in mice."}},"tag":"EXP"},{"id":281,"details":{"paperId":"7c8fb2d61034bbe7ae3f450675ae63edcae3e73d","externalIds":{"MAG":"2345488562","DOI":"10.1128/JVI.00423-16","CorpusId":"4782274","PubMed":"27147743"},"title":"Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. IMPORTANCE RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development and modifying essential NS2 structural features to marginally limit viral growth while maintaining immune responses."}},"tag":"EXP"},{"id":377,"details":{"paperId":"7190525678378e3c01322d31465ef49351dfb9a8","externalIds":{"MAG":"2115225611","DOI":"10.1128/JVI.76.6.2871-2880.2002","CorpusId":"36774058","PubMed":"11861854"},"title":"Identification of Temperature-Sensitive Mutations in the Phosphoprotein of Respiratory Syncytial Virus That Are Likely Involved in Its Interaction with the Nucleoprotein","abstract":"ABSTRACT The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37°C in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39°C. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33°C, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37°C. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37°C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro, and it was demonstrated that N-P interaction is impaired at 37°C."}},"tag":"EXP"},{"id":359,"details":{"paperId":"cd4a8550afd36e979f5b83cb78b1f9f9b40192c5","externalIds":{"MAG":"2105952979","DOI":"10.1128/JVI.73.8.6610-6617.1999","CorpusId":"22141214","PubMed":"10400758"},"title":"Identification of a Linear Heparin Binding Domain for Human Respiratory Syncytial Virus Attachment Glycoprotein G","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children worldwide. Infection is mediated, in part, by an initial interaction between attachment protein (G) and a highly sulfated heparin-like glycosaminoglycan (Gag) located on the cell surface. Synthetic overlapping peptides derived from consensus sequences of the G protein ectodomain from both RSV subgroups A and B were tested by heparin-agarose affinity chromatography for their abilities to bind heparin. This evaluation identified a single linear heparin binding domain (HBD) for RSV subgroup A (184A→T198) and B (183K→K197). The binding of these peptides to Vero cells was inhibited by heparin. Peptide binding to two CHO cell mutants (pgsD-677 and pgsA-745) deficient in heparan sulfate or total Gag synthesis was decreased 50% versus the parental cell line, CHO-K1, and decreased an average of 87% in the presence of heparin. The RSV-G HBD peptides were also able to inhibit homologous and heterologous virus infectivity of Vero cells. These results indicate that the sequence184A/183K→198T/K197for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results indicate that the sequence184A/183K→198T/K197for RSV subgroups A and B, respectively, defines an important determinant of RSV-G interactions with heparin."}},"tag":"EXP"},{"id":379,"details":{"paperId":"610d5f6f32cb73d775aee359260fe6ae99273098","externalIds":{"MAG":"2124023078","DOI":"10.1128/JVI.77.13.7352-7360.2003","CorpusId":"6681173","PubMed":"12805433"},"title":"Identification of a Single Amino Acid Change in the Human Respiratory Syncytial Virus L Protein That Affects Transcriptional Termination","abstract":"ABSTRACT Infectious Human respiratory syncytial virus (HRSV) with an aberrant RNA synthesis pattern was recovered from a cDNA clone. The virus displayed increased levels of polycistronic readthrough mRNAs resulting from failure of the polymerase to terminate transcription efficiently at the gene ends. An asparagine (N) to aspartic acid (D) change at amino acid 1049 in the large (L) polymerase protein was found to be responsible for the readthrough phenotype. Virus encoding N at position 1049 displayed an RNA synthesis pattern indistinguishable from the A2 strain of HRSV. We compared the transcriptional activities of polymerases that encoded an N or D at position 1049 (L1049N or L1049D) by using dicistronic subgenomic replicons and found that the alteration of amino acid 1049 specifically affected transcriptional termination but had no effect on genome replication. L1049N recognized and terminated transcription at each of the naturally occurring gene junctions with differing efficiencies but at significantly higher efficiency than L1049D. The abilities of the polymerases to recognize the cis-acting gene end signals required for termination were compared by examining the effect of single nucleotide changes at positions 1 to 8 of the M gene end and were found to be similar. This work identifies a single amino acid position in the polymerase that specifically affects the ability of the polymerase to terminate transcription, but which does not affect genome replication or interaction with the M2-1 protein. This work shows the critical importance of the previously defined cis-acting signals for termination, confirms differential termination at the varied gene junctions, and shows that the polymerase responds to the cis-acting sequences similarly, whether it has N or D at position 1049.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The critical importance of the previously defined cis-acting signals for termination is shown, differential termination at the varied gene junctions is confirmed, and the polymerase responds to the cis- acting sequences similarly, whether it has N or D at position 1049."}},"tag":"EXP"},{"id":95,"details":{"paperId":"b45dd7f4512b385fd4aea9b772cb4850cba35afd","externalIds":{"MAG":"2940374012","DOI":"10.1016/j.virol.2019.04.001","CorpusId":"128361934","PubMed":"31009855"},"title":"Identification of a human respiratory syncytial virus phosphoprotein domain required for virus-like-particle formation.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data show that P not only co-regulates replication and transcription but also has an important role in assembly, mediated by a separate domain that likely interacts with M and/or F and is highly regulated by phosphorylation."}},"tag":"EXP"},{"id":222,"details":{"paperId":"997734275ff151fc985771c0fc4ef4bf65b7c4b3","externalIds":{"MAG":"2022246526","DOI":"10.1099/0022-1317-75-3-555","CorpusId":"26036973","PubMed":"8126452"},"title":"Identification of a protein kinase involved in the phosphorylation of the C-terminal region of human respiratory syncytial virus P protein.","abstract":"P protein, the structural phosphoprotein of the Long strain of respiratory syncytial (RS) virus, is phosphorylated at serine residues. Some of these residues are candidates for modification by casein kinase II, as they are contained in consensus sequences. A cellular protein kinase, able to phosphorylate the P protein in vitro and apparently associated with purified RS virions, has been partially purified from HEp-2 cells. It shows several characteristics similar to those of casein kinase II. The P protein is modified in vitro by this activity mainly at serine residues located near the C terminus, which are also modified during virus infection. Thus, the P protein is phosphorylated in vivo in two regions, a central region as previously described, and another located in the C-terminal part of the molecule. The protein kinase involved in the phosphorylation of the C-terminal domain is similar to a cellular casein kinase II.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A cellular protein kinase, able to phosphorylate the P protein in vitro and apparently associated with purified RS virions, has been partially purified from HEp-2 cells and shows several characteristics similar to those of casein kinase II."}},"tag":"EXP"},{"id":136,"details":{"paperId":"f12e5bdbdc2ab01cae1b15934948989407a2d72c","externalIds":{"MAG":"2053347879","DOI":"10.1038/nm.2444","CorpusId":"205388029","PubMed":"21841784"},"title":"Identification of nucleolin as a cellular receptor for human respiratory syncytial virus","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV interacts with host-cell nucleolin via the viral fusion envelope glycoprotein and binds specifically to nucleolin at the apical cell surface in vitro and RNAi-mediated knockdown of lung nucleolin was associated with a significant reduction in RSV infection in mice, confirming that nucleolin is a functional RSV receptor in vivo."}},"tag":"EXP"},{"id":424,"details":{"paperId":"e88dd66b9a56f99cce3fcdcef1028935d791dfa6","externalIds":{"PubMedCentral":"7796524","DOI":"10.1186/s12887-020-02480-4","CorpusId":"231202019","PubMed":"33419394"},"title":"Identification of pathogenic genes and transcription factors in respiratory syncytial virus","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The expression of IFI27, IFI44, IFITM3, FCER1A, and ISG15 were shown to be involved in the pathogenesis of RSV, and this finding may contribute to the development of new potential biomarkers, reveal the underlying pathogenesis and also identify novel therapeutic targets for RSV."}},"tag":"EXP"},{"id":343,"details":{"paperId":"ef1607b5d11710c809d416e5e932344850eb0396","externalIds":{"MAG":"2167021975","DOI":"10.1128/jvi.70.2.801-808.1996","CorpusId":"6636245","PubMed":"8551618"},"title":"Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions","abstract":"We have reported previously that the nucleoprotein (N), the phosphoprotein (P), and the 22-kDa protein of human respiratory syncytial virus (HRSV) are components of the cytoplasmic inclusion bodies observed in HEp-2-infected cells. In addition, coexpression of N and P was sufficient to induce the formation of N-P complexes detectable by either coimmunoprecipitation with anti-P antibodies or generation of cytoplasmic inclusions. We now report the identification of protein regions required for these interactions. Deletion mutant analysis of the P protein gene indicated that its C-terminal end was essential for interacting with N. This conclusion was strengthened by the finding that an anti-P monoclonal antibody (021/12P), reacting with a 21-residue P protein C-terminal peptide, apparently displaced N from N-P complexes. The same effect was observed with high concentrations of the C-terminal peptide. However, sequence requirements for the P protein C-terminal end were not absolute, and mutants with the substitution Ser-237-->Ala or Ser-237-->Thr were as efficient as the wild type in interacting with N. In addition, P and N proteins from strains of different HRSV antigenic groups, with sequence differences in the P protein C-terminal end, were able to coimmunoprecipitate and formed cytoplasmic inclusions. Deletion mutant analysis of the N gene indicated that large segments of this polypeptide were required for interacting with P. The relevance of these interactions for HRSV is discussed in comparison with those of analogous proteins from related viruses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"P and N proteins from strains of different HRSV antigenic groups, with sequence differences in the P protein C-terminal end, were able to coimmunoprecipitate and formed cytoplasmic inclusions and the relevance of these interactions for H RSV is discussed in comparison with those of analogous proteins from related viruses."}},"tag":"EXP"},{"id":352,"details":{"paperId":"fe1e99eae96c98d6042454c68de43b88cba8e4c6","externalIds":{"MAG":"2105379708","DOI":"10.1128/JVI.72.7.5707-5716.1998","CorpusId":"24113675","PubMed":"9621029"},"title":"Identification of the Respiratory Syncytial Virus Proteins Required for Formation and Passage of Helper-Dependent Infectious Particles","abstract":"ABSTRACT We developed a system to identify the viral proteins required for the packaging and passage of human respiratory syncytial virus (RSV) by reconstructing these events with cDNA-encoded components. Plasmids encoding individual RSV proteins, each under the control of a T7 promoter, were cotransfected in various combinations together with a plasmid containing a minigenome into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Supernatants from these cells were passaged onto fresh cells which were then superinfected with RSV. Functional reconstitution of RSV-specific packaging and passage was detected by expression of the reporter gene carried on the minigenome. As expected, the four nucleocapsid proteins N, P, L, and M2-1 failed to direct packaging and passage of the minigenome. Passage was achieved by further addition of plasmids expressing three membrane-associated proteins, M, G, and F; inclusion of the fourth envelope- associated protein, SH, did not alter passage efficiency. Passage was reduced 10- to 20-fold by omission of G and was abrogated by omission of either M or F. Coexpression of the nonstructural NS1 or NS2 protein had little effect on packaging and passage except through indirect effects on RNA synthesis in the initial transfection. The M2-1 transcription elongation factor was not required for the generation of passage-competent particles. However, addition of increasing quantities of M2-1 to the transfection mediated a dose-dependent inhibition of passage which was alleviated by coexpression of the putative negative regulatory factor M2-2. Omission of the L plasmid reduced passage 10- to 20-fold, most likely due to reduced availability of encapsidated minigenomes for packaging. However, the residual level of passage indicated that neither L protein nor the process of RSV-specific RNA synthesis is required for the production and passage of particles. Omission of N or P from the transfection abrogated passage. Thus, the minimum RSV protein requirements for packaging and passaging a minigenome are N, P, M, and F, although the efficiency is greatly increased by addition of L and G.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The residual level of passage indicated that neither L protein nor the process of RSV-specific RNA synthesis is required for the production and passage of particles, and the minimum RSV protein requirements for packaging and passaging a minigenome are N, P, M, and F, although the efficiency is greatly increased by addition of L and G."}},"tag":"EXP"},{"id":232,"details":{"paperId":"99baf342dc92353180251339a77c9f96e2fb2fca","externalIds":{"MAG":"2734855606","DOI":"10.1099/jgv.0.000852","CorpusId":"2460266","PubMed":"28708049"},"title":"Identification of two novel functional tRNA-derived fragments induced in response to respiratory syncytial virus infection.","abstract":"Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection (LRTI) in children from infancy up to early childhood. Recently, we demonstrated that RSV infection alters cellular small non-coding RNA (sncRNA) expression, most notably the tRNA-derived RNA fragments (tRFs). However, the functions of the tRFs in virus-host interaction are largely unknown. Herein, we examined the role of three RSV-induced tRFs derived from the 5-end of mature tRNAs decoding GlyCCC, LysCTT and CysGCA (named tRF5-GlyCCC, tRF5-LysCTT and tRF5-CysGCA, respectively) in controlling RSV replication. We found that tRF5-GlyCCC and tRF5-LysCTT, but not tRF5-CysGCA, promote RSV replication, demonstrating the functional specificity of tRFs. The associated molecular mechanisms underlying the functions of tRF5-GlyCCC and tRF5-LysCTT were also investigated. Regulating the expression and/or activity of these tRFs may provide new insights into preventive and therapeutic strategies for RSV infection. The study also accumulated data for future development of a tRF targeting algorithm.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This study examined the role of three RSV-induced tRFs derived from the 5-end of mature tRNAs decoding GlyCCC, LysCTT and CysGCA in controlling RSV replication and found that tRF5-GlyCCC and t RF5-LysCTT, but not tRF 5-CysG CA, promote RSV replicate, demonstrating the functional specificity of t RFs."}},"tag":"EXP"},{"id":217,"details":{"paperId":"23937e52a5288478d0037f21b054794bdfe8b6e2","externalIds":{"MAG":"2141154963","DOI":"10.1099/0022-1317-72-9-2091","CorpusId":"15453833","PubMed":"1895054"},"title":"Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses.","abstract":"We have previously classified isolates from a respiratory syncytial (RS) virus epidemic into distinct lineages by restriction mapping and nucleotide sequencing of parts of the nucleocapsid protein and small hydrophobic protein genes, which are areas of the genome not considered to be under immunological pressure. This study has now been extended by the determination of the nucleotide sequences of the attachment (G) protein genes of isolates from each subgroup A lineage. Deduced amino acid identities of the G proteins ranged between 80% and 99%, corresponding closely to the previously determined relatedness of the lineages. The amino acid variability was not evenly distributed; in the extracellular part of the protein there was a sharply defined hypervariable domain which was separated from a more extended variable domain by a highly conserved region. Most nucleotide changes in the variable domains were in the first and second positions of the codon triplets. These results suggest that there may be considerable immunological pressure for change in certain areas of the G protein and this may account for the ability of this virus to reinfect individuals repeatedly. The results presented here reflect the pattern of published data comparing prototype strains of the A and B subgroups.","publicationTypes":["JournalArticle","Study"],"tldr":{"model":"tldr@v2.0.0","text":"The results suggest that there may be considerable immunological pressure for change in certain areas of the G protein and this may account for the ability of this virus to reinfect individuals repeatedly."}},"tag":"EXP"},{"id":18,"details":{"paperId":"4e2672db49ef3ade0226eed0ebcd6249509d17e7","externalIds":{"MAG":"1972873370","DOI":"10.1006/VIRO.2000.0293","CorpusId":"11626777","PubMed":"10860881"},"title":"Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection.","abstract":"Respiratory syncytial virus (RSV) is an important human respiratory pathogen, particularly in infants. Glycosaminoglycans (GAGs) have been implicated in the initiation of RSV infection of cultured cells, but it is not clear what type of GAGs and GAG components are involved, whether the important GAGs are on the virus or the cell, or what the magnitude is of their contribution to infection. We constructed and rescued a recombinant green fluorescent protein (GFP)-expressing RSV (rgRSV) and used this virus to develop a sensitive system to assess and quantify infection by flow cytometry. Evaluation of a panel of mutant Chinese hamster ovary cell lines that are genetically deficient in various aspects of GAG synthesis showed that infection was reduced up to 80% depending on the type of GAG deficiency. Enzymatic removal of heparan sulfate and/or chondroitin sulfate from the surface of HEp-2 cells also reduced infection, and the removal of both reduced infection even further. Blocking experiments in which RSV was preincubated with various soluble GAGs revealed the relative blocking order of: heparin > heparan sulfate > chondroitin sulfate B. Iduronic acid is a component common to these GAGs. GAGs that do not contain iduronic acid, namely, chondroitin sulfate A and C and hyaluronic acid, did not inhibit infection. A role for iduronic acid-containing GAGs in RSV infection was confirmed by the ability of basic fibroblast growth factor to block infection, because basic fibroblast growth factor binds to GAGs containing iduronic acid. Pretreatment of cells with protamine sulfate, which binds and blocks GAGs, also reduced infection. In these examples, infection was reduced by pretreatment of the virus with soluble GAGs, pretreatment of the cells with GAG-binding molecules, pretreatment of the cells with GAG-destroying enzymes or in cells genetically deficient in GAGs. These results establish that the GAGs involved in RSV infection are present on the cell rather than on the virus particle. Thus, the presence of cell surface GAGs containing iduronic acid, like heparan sulfate and chondroitin sulfate B, is required for efficient RSV infection in cell culture.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is established that the GAGs involved in RSV infection are present on the cell rather than on the virus particle, and the presence of cell surface G AGs containing iduronic acid, like heparan sulfate and chondroitin sulfate B, is required for efficient RSV infections in cell culture."}},"tag":"EXP"},{"id":482,"details":{"paperId":"b26613ba33656b3ed67c157dd9eedba69c5e00cc","externalIds":{"PubMedCentral":"6478035","MAG":"2939517022","DOI":"10.3389/fimmu.2019.00810","CorpusId":"115205493","PubMed":"31057543"},"title":"Immune-Modulation by the Human Respiratory Syncytial Virus: Focus on Dendritic Cells","abstract":"The human respiratory syncytial virus (hRSV) is the leading cause of pneumonia in infants and produces a significant burden in the elderly. It can also infect and produce disease in otherwise healthy adults and recurrently infect those previously exposed to the virus. Importantly, recurrent infections are not necessarily a consequence of antigenic variability, as described for other respiratory viruses, but most likely due to the capacity of this virus to interfere with the host's immune response and the establishment of a protective and long-lasting immunity. Although some genes encoded by hRSV are known to have a direct participation in immune evasion, it seems that repeated infection is mainly given by its capacity to modulate immune components in such a way to promote non-optimal antiviral responses in the host. Importantly, hRSV is known to interfere with dendritic cell (DC) function, which are key cells involved in establishing and regulating protective virus-specific immunity. Notably, hRSV infects DCs, alters their maturation, migration to lymph nodes and their capacity to activate virus-specific T cells, which likely impacts the host antiviral response against this virus. Here, we review and discuss the most important and recent findings related to DC modulation by hRSV, which might be at the basis of recurrent infections in previously infected individuals and hRSV-induced disease. A focus on the interaction between DCs and hRSV will likely contribute to the development of effective prophylactic and antiviral strategies against this virus.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The most important and recent findings related to DC modulation by hRSV are reviewed, which might be at the basis of recurrent infections in previously infected individuals and h RSV-induced disease."}},"tag":"EXP"},{"id":408,"details":{"paperId":"150d4adf7abc884d82e29767601783b9dc747a8f","externalIds":{"MAG":"3013722060","DOI":"10.1152/physrev.00030.2019","CorpusId":"214679754","PubMed":"32216549"},"title":"Impact of Respiratory Syncytial Virus Infection on the Host Cell: Implications for Antiviral Strategies.","abstract":"Respiratory syncytial virus (RSV) is one of the leading causes of viral respiratory tract infection in infants, the elderly and the immunocompromised worldwide, causing more deaths each year than influenza. Years of research into RSV since its discovery over 60 years ago have elucidated detailed mechanisms of the host-pathogen interface. RSV infection elicits widespread transcriptomic and proteomic changes, which both mediate the host innate and adaptive immune responses to infection, and reflect RSV's ability to circumvent the host stress responses, including stress granule formation, endoplasmic reticulum stress, oxidative stress, and programmed cell death. The combination of these events can severely impact on human lungs, resulting in airway remodeling and pathophysiology. The RSV membrane envelope glycoproteins (fusion F and attachment G), matrix (M) and nonstructural (NS) 1 and 2 proteins play key roles in modulating host cell functions to promote the infectious cycle. This review presents a comprehensive overview of how RSV impacts the host response to infection, and how detailed knowledge of the mechanisms thereof can inform the development of new approaches to develop RSV vaccines and therapeutics.","publicationTypes":["JournalArticle","Review"],"tldr":{"model":"tldr@v2.0.0","text":"This review presents a comprehensive overview of how RSV impacts the host response to infection, and how detailed knowledge of the mechanisms thereof can inform the development of new approaches to develop RSV vaccines and therapeutics."}},"tag":"EXP"},{"id":68,"details":{"paperId":"7a881b2cf4f0178635380a1daecded3b1329bab9","externalIds":{"PubMedCentral":"9436823","DOI":"10.1016/j.jbc.2022.102337","CorpusId":"251328202","PubMed":"35931116"},"title":"Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5′ end of RNA does not explain the specificity of encapsidation, and showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories."}},"tag":"EXP"},{"id":310,"details":{"paperId":"acde1ebf06b8bef489b3f9e0e516ccc7f6433592","externalIds":{"MAG":"3092205204","PubMedCentral":"7737744","DOI":"10.1128/JVI.01897-20","CorpusId":"222217614","PubMed":"33028717"},"title":"In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus","abstract":"As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase. ABSTRACT Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis. IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis, and the sequence specificity of different viral promoters was compared and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV Polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay."}},"tag":"EXP"},{"id":181,"details":{"paperId":"04a6de9b358caa9c073cc46da196fd4ce18865b4","externalIds":{"MAG":"2995675501","PubMedCentral":"6970927","DOI":"10.1074/jbc.RA119.011602","CorpusId":"209316780","PubMed":"31822560"},"title":"In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus","abstract":"The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This work established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs, discovering that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NclPs."}},"tag":"EXP"},{"id":14,"details":{"paperId":"2f92bba318030f6251b7a61deae4fead67469589","externalIds":{"MAG":"2078768973","DOI":"10.1006/VIRO.1997.8734","CorpusId":"38876499","PubMed":"9299631"},"title":"Increased expression of the N protein of respiratory syncytial virus stimulates minigenome replication but does not alter the balance between the synthesis of mRNA and antigenome.","abstract":"A popular model for RNA synthesis by nonsegmented negative-strand RNA viruses is that transcription and RNA replication are executed by the same polymerase complex and that there is a dynamic balance between the two processes that is mediated by the nucleocapsid N protein. According to this model, transcription occurs until sufficient soluble N protein accumulates to initiate encapsidation of the nascent RNA product, which somehow switches the polymerase into a readthrough replicative mode. This model was examined for respiratory syncytial virus (RSV) using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins that are expressed intracellularly from transfected plasmids. Preliminary experiments showed that reconstituted RNA replication was highly productive, such that on average each molecule of plasmid-supplied minigenome that became encapsidated was amplified 10- to 50-fold. N protein was increased on its own or in concert with the phosphoprotein P and in the presence or absence of the M2 ORF1 transcription elongation factor. The maximum level of N and P protein expression achieved from plasmids equalled or exceeded that obtained in RSV-infected cells. Increased levels of N protein stimulated RNA replication. This is consistent with the idea that RNA replication is dependent on the availability of N protein for encapsidation, which is one postulate of the model. The M2 ORF1 protein had no detectable effect on RNA replication under the various conditions of expression of N and P, which confirmed and extended previous results. However, there was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (synthesis of antigenome). The synthesis of positive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"There was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (syndhesis of antigenome), and the synthesis ofpositive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product."}},"tag":"EXP"},{"id":54,"details":{"paperId":"cd0c979c911b24a3412e2cd5c9ab807c69894feb","externalIds":{"MAG":"1974788227","DOI":"10.1016/j.antiviral.2013.08.012","CorpusId":"41536185","PubMed":"23994498"},"title":"Increased hydroxymethylglutaryl coenzyme A reductase activity during respiratory syncytial virus infection mediates actin dependent inter-cellular virus transmission.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Investigation of the role that hydroxymethylglutaryl coenzyme A reductase (HMGCR) plays during respiratory syncytial virus (RSV) maturation suggested that HMGCR-mediated changes in F-actin structure play an important role in the inter-cellular transmission of mature RSV particles."}},"tag":"EXP"},{"id":344,"details":{"paperId":"80ce916eb2243eacac2f2866bfd944019a281399","externalIds":{"MAG":"1780848409","DOI":"10.1128/jvi.70.3.1554-1563.1996","CorpusId":"19773094","PubMed":"8627674"},"title":"Inducible translational regulation of the NF-IL6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells","abstract":"Respiratory syncytial virus (RSV), the most common etiologic agent of epidemic pediatric respiratory disease, infects and replicates in the human airway epithelium, resulting in the induction of cellular gene products essential for immune and inflammatory responses. We describe the effect of RSV infection on nuclear factor-IL6 (NF-IL6) expression, a human basic domain-leucine zipper-containing transcription factor that alone in combination with other inducible transcription factors regulates the expression of cytokine and adhesion molecule genes. RSV-infected human type II pulmonary alveolar epithelial cells (A549) synthesize a single 45.7-kDa isoform of NF-IL6 rapidly and in a time-dependent manner. NF-IL6 is first detectable after 3 h of infection and continues to accumulate until 48 h (until the cells lose viability). NF-IL6 production could not be induced by UV-inactivated virus, demonstrating the requirement of viral replication for NF-IL6 synthesis. Immunoprecipitation after [35S]methionine metabolic labeling was done to investigate the mechanism for NF-IL6 production. There was robust NF-IL6 protein synthesis within RSV-infected (24 h) cells. Protein synthesis occurred without detectable changes in the abundance or size of the single 1.8-kb NF-IL6 mRNA. RNase protection assay of transfected chloramphenicol acetyltransferase reporter genes driven by either wild-type or mutated NF-IL6 binding sites show a virus-induced increase in NF-IL6-dependent transcription. These studies have demonstrated a novel inducible mechanism for translational control of NF-IL6 synthesis and identify this transcription factor as a potential effector of the host response to RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A novel inducible mechanism for translational control of NF-IL6 synthesis is demonstrated and this transcription factor is identified as a potential effector of the host response to RSV infection."}},"tag":"EXP"},{"id":188,"details":{"paperId":"ef247eff6f797dc22a6e068b584f3243daf7f191","externalIds":{"MAG":"2311703219","DOI":"10.1080/21505594.2016.1144001","CorpusId":"205957623","PubMed":"26809688"},"title":"Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus","abstract":"ABSTRACT Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress, and suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of H RSV infections."}},"tag":"EXP"},{"id":87,"details":{"paperId":"f851063fa1a21f733dad3f9ceb28f9522563e125","externalIds":{"MAG":"2008997405","DOI":"10.1016/j.virol.2008.11.043","CorpusId":"205642802","PubMed":"19128816"},"title":"Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This work compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC), and used GFP-expressing viruses to identify robustly infected cells and distinguish between direct effects of robust viral gene expression versus bystander effects."}},"tag":"EXP"},{"id":195,"details":{"paperId":"ff34f26aea71f082f8e689d4f4702b657f277767","externalIds":{"MAG":"2316632082","DOI":"10.1093/INFDIS/165.5.819","CorpusId":"125504","PubMed":"1373752"},"title":"Infectious respiratory syncytial virus (RSV) effectively inhibits the proliferative T cell response to inactivated RSV in vitro.","abstract":"The effect of respiratory syncytial virus (RSV) on the cellular immune response of human mononuclear cells in vitro was examined. Inhibition by RSV of the lymphocyte response to phytohemagglutinin in vitro was confirmed using cells from human umbilical cord blood. In addition, RSV significantly inhibited both the proliferative and T cell colony responses of human mononuclear cells to Epstein-Barr virus. An RSV-specific cellular immune response was induced in vitro by stimulation of mononuclear cells from RSV-seropositive donors with beta-propiolactone-inactivated RSV. This RSV-specific response was significantly inhibited by infectious RSV itself, and the inhibition was mediated by an extracellular factor produced by RSV-infected mononuclear cells. A similar inhibition in vivo of the RSV-induced cellular immune response may contribute significantly to delayed recovery from primary infection and to reduced resistance to subsequent infections.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Inhibition by RSV of the lymphocyte response to phytohemagglutinin in vitro was confirmed using cells from human umbilical cord blood, and RSV significantly inhibited both the proliferative and T cell colony responses of human mononuclear cells to Epstein-Barr virus."}},"tag":"EXP"},{"id":130,"details":{"paperId":"5eaef675e3c6f8edac7858130b5b92286abe32f7","externalIds":{"MAG":"2075445533","DOI":"10.1021/la104041n","CorpusId":"7324748","PubMed":"21141948"},"title":"Influence of lipids on the interfacial disposition of respiratory syncytical virus matrix protein.","abstract":"The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers. This has enabled factors that influence the disposition of the protein with respect to the lipid interface to be characterized. In the absence of sphingomyelin, respiratory syncytial virus matrix protein penetrates monolayers composed of mixtures of phosphocholines with phosphoethanolamines or cholesterol at the air-water interface. In ternary mixtures composed of sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol, the protein exhibits two separate behaviors: (1) peripheral association with the surface of sphingomyelin-rich domains and (2) penetration of sphingomyelin-poor domains. Prolonged incubation of the protein with mixtures of phosphocholines and phosphoethanolamines leads to the formation of helical protein assemblies of uniform diameter that demonstrate an inherent propensity of the protein to assemble into a filamentous form.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscope on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers."}},"tag":"EXP"},{"id":38,"details":{"paperId":"b4bdffc611e73e2b9d43439b9a24724b9a4abf86","externalIds":{"DOI":"10.1007/s00705-021-04974-7","CorpusId":"231910537","PubMed":"33580381"},"title":"Inhibition of AMP-activated protein kinase in respiratory syncytial virus infection activates lipid metabolism","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data show that RSV infection inhibits AMPK activity, favoring the activation of downstream lipogenic effectors and cellular lipid anabolism in HEp-2 cells."}},"tag":"EXP"},{"id":264,"details":{"paperId":"01fce97ddd5dfee29fa2912b72b1ae8c652e342b","externalIds":{"MAG":"1564985569","DOI":"10.1111/j.1749-6632.2010.05560.x","CorpusId":"22807316","PubMed":"20716287"},"title":"Inhibition of epithelial sodium channels by respiratory syncytial virus in vitro and in vivo","abstract":"Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and children worldwide. Infection of mice with RSV decreased sodium (Na+) dependent alveolar fluid clearance (AFC), resulting in increased lung water content and hypoxemia. RSV infection resulted in higher levels of pyrimidines and purines in the alveolar space. Intratracheal administration of UTP or UDP also decreased AFC. The effects of RSV on AFC and oxygen saturation of Balb/c mice were reversed by intraalveolar administration of antagonists of P2Y nucleotide receptors, enzymes that enhance the breakdown of pyrimidines and systemic or intranasal administration of inhibitors of the de novo pathway of pyrimidine synthesis. RSV infection of H441 or mouse tracheal epithelial cells decreased the amiloride‐sensitive Na+ currents and pretreatment of H441 cells with A77 prevented this effect. These findings indicate that the harmful effects of RSV on lung epithelia are mediated at least in part via the production of UTP and its paracrine action on ENaC.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Findings indicate that the harmful effects of RSV on lung epithelia are mediated at least in part via the production of UTP and its paracrine action on ENaC."}},"tag":"EXP"},{"id":290,"details":{"paperId":"54de726db9eb9c94f1d57cbb125295110a9f9c55","externalIds":{"MAG":"2098918223","DOI":"10.1128/JVI.00839-14","CorpusId":"43601092","PubMed":"25100835"},"title":"Inhibition of the Human Respiratory Syncytial Virus Small Hydrophobic Protein and Structural Variations in a Bicelle Environment","abstract":"ABSTRACT The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. IMPORTANCE The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The first inhibitor of the SH protein channel is reported, pyronin B, and its binding site is mapped to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain."}},"tag":"EXP"},{"id":266,"details":{"paperId":"69168d66bc1de37bc1430fcae7d11050967a220d","externalIds":{"MAG":"3043973192","DOI":"10.1128/AAC.00608-20","CorpusId":"220841797","PubMed":"32718963"},"title":"Inhibitory Effect of PIK-24 on Respiratory Syncytial Virus Entry by Blocking Phosphatidylinositol-3 Kinase Signaling","abstract":"Phosphoinositide-3 kinase signaling modulates many cellular processes, including cell survival, proliferation, differentiation, and apoptosis. Currently, it is known that the establishment of respiratory syncytial virus infection requires phosphoinositide-3 kinase signaling. However, the regulatory pattern of phosphoinositide-3 kinase signaling or its corresponding molecular mechanism during respiratory syncytial virus entry remains unclear. Here, the involvement of phosphoinositide-3 kinase signaling in respiratory syncytial virus entry was studied. ABSTRACT Phosphoinositide-3 kinase signaling modulates many cellular processes, including cell survival, proliferation, differentiation, and apoptosis. Currently, it is known that the establishment of respiratory syncytial virus infection requires phosphoinositide-3 kinase signaling. However, the regulatory pattern of phosphoinositide-3 kinase signaling or its corresponding molecular mechanism during respiratory syncytial virus entry remains unclear. Here, the involvement of phosphoinositide-3 kinase signaling in respiratory syncytial virus entry was studied. PIK-24, a novel compound designed with phosphoinositide-3 kinase as a target, had potent anti-respiratory syncytial virus activity both in vitro and in vivo. PIK-24 significantly reduced viral entry into the host cell through blocking the late stage of the fusion process. In a mouse model, PIK-24 effectively reduced the viral load and alleviated inflammation in lung tissue. Subsequent studies on the antiviral mechanism of PIK-24 revealed that viral entry was accompanied by phosphoinositide-3 kinase signaling activation, downstream RhoA and cofilin upregulation, and actin cytoskeleton rearrangement. PIK-24 treatment significantly reversed all these effects. The disruption of actin cytoskeleton dynamics or the modulation of phosphoinositide-3 kinase activity by knockdown also affected viral entry efficacy. Altogether, it is reasonable to conclude that the antiviral activity of PIK-24 depends on the phosphoinositide-3 kinase signaling and that the use of phosphoinositide-3 kinase signaling to regulate actin cytoskeleton rearrangement plays a key role in respiratory syncytial virus entry.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"PIK-24, a novel compound designed with phosphoinositide-3 kinase as a target, had potent anti-respiratory syncytial virus activity both in vitro and in vivo and it is reasonable to conclude that the antiviral activity of Pik-24 depends on the phosphoinoskeleton signaling."}},"tag":"EXP"},{"id":175,"details":{"paperId":"f1f3ce4bd284b507d8c96d441aba04c825b1ad65","externalIds":{"MAG":"2011869970","DOI":"10.1074/jbc.M113.510594","CorpusId":"26541644","PubMed":"24497642"},"title":"Inositol-requiring Enzyme 1 Inhibits Respiratory Syncytial Virus Replication*","abstract":"Background: The endoplasmic reticulum (ER) stress response is increasingly implicated in the pathogenesis of viral infections. Results: Respiratory syncytial virus (RSV) infection induces the inositol-requiring enzyme 1 (IRE1) stress pathway. IRE1 activity inhibits RSV replication. Conclusion: ER stress may be a novel cellular anti-RSV defense mechanism. Significance: Identification of a novel regulator of RSV replication may have therapeutic implications. Despite being a major health problem, respiratory syncytial virus (RSV) infections remain without specific therapy. Identification of novel host cellular responses that play a role in the pathogenesis of RSV infection is needed for therapeutic development. The endoplasmic reticulum (ER) stress response is an evolutionarily conserved cellular signaling cascade that has been implicated in multiple biological phenomena, including the pathogenesis of some viral infections. In this study, we investigate the role of the ER stress response in RSV infection using an in vitro A549 cell culture model. We found that RSV infection induces a non-canonical ER stress response with preferential activation of the inositol-requiring enzyme 1 (IRE1) and activated transcription factor 6 (ATF6) pathways with no concomitant significant activation of the protein kinase R-like ER kinase (PERK) pathway. Furthermore, we discovered that IRE1 has an inhibitory effect on RSV replication. Our data characterize, for the first time, the nature of the ER stress response in the setting of RSV infection and identify the IRE1 stress pathway as a novel cellular anti-RSV defense mechanism.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data characterize, for the first time, the nature of the ER stress response in the setting of RSV infection and identify the IRE1 stress pathway as a novel cellular anti-RSV defense mechanism."}},"tag":"EXP"},{"id":380,"details":{"paperId":"1248eda7b3c7236be4de97495ae591a1390a38d5","externalIds":{"MAG":"2137165958","DOI":"10.1128/JVI.77.19.10670-10676.2003","CorpusId":"12445418","PubMed":"12970453"},"title":"Interaction between Human Respiratory Syncytial Virus (RSV) M2-1 and P Proteins Is Required for Reconstitution of M2-1-Dependent RSV Minigenome Activity","abstract":"ABSTRACT We have investigated protein-protein interactions among the respiratory syncytial virus (RSV) RNA polymerase subunits using affinity chromatography. Here we demonstrate a novel interaction of P and M2-1 proteins. Phosphorylation of either M2-1 or P appears to be dispensable for this interaction. Internal deletions within P mapped the M2-1-binding domain to a region between residues 100 and 120. Alanine-scanning mutagenesis within this region of P revealed that substitution of any one of the three residues, L101, Y102, and F109, prevented both M2-1 and P binding and expression of an M2-1-dependent luciferase reporter gene. However, these same mutations did not prevent the activity of an M2-1-independent chloramphenicol acetyltransferase minigenome, suggesting that these residues of P specifically affect M2-1-P interaction. On the basis of these observations, it is possible that the interaction between RSV M2-1 and P proteins is important for viral replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A novel interaction of P and M2-1 proteins is demonstrated, showing that substitution of any one of the three residues, L101, Y102, and F109, prevented both M 2-1 and P binding and expression of an M1-1-dependent luciferase reporter gene."}},"tag":"EXP"},{"id":93,"details":{"paperId":"4d6282f79d1875152f6a3090d899a97142355d95","externalIds":{"MAG":"2077558186","DOI":"10.1016/j.virol.2015.03.034","CorpusId":"205651186","PubMed":"25854864"},"title":"Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A membrane-based yeast two-hybrid system and a library from human lung cDNA were used to detect proteins that bind SH protein, leading to the identification of a membrane protein, B-cell associated protein 31 (BAP31), which may constitute a potential drug target."}},"tag":"EXP"},{"id":26,"details":{"paperId":"14e393f09feea6fb5dc01c7c34e729d442acf62c","externalIds":{"MAG":"2413594285","DOI":"10.1007/978-1-59745-393-6_2","CorpusId":"34807641","PubMed":"17502668"},"title":"Interaction between respiratory syncytial virus and glycosaminoglycans, including heparan sulfate.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The rationale and methods for analyzing GAG usage by one such virus, respiratory syncytial virus (RSV), are described and a method for rapidly determining whether G AG usage is characteristic of a wild virus or is limited to laboratory-adapted virus is presented."}},"tag":"EXP"},{"id":244,"details":{"paperId":"940bd4a1dc7c09838686b611f1a550f25a9fa75b","externalIds":{"MAG":"1976627514","DOI":"10.1099/VIR.0.80829-0","CorpusId":"5775605","PubMed":"15958665"},"title":"Interaction between the respiratory syncytial virus G glycoprotein cytoplasmic domain and the matrix protein.","abstract":"Paramyxovirus assembly at the cell membrane requires the movement of viral components to budding sites and envelopment of nucleocapsids by cellular membranes containing viral glycoproteins, facilitated by interactions with the matrix protein. The specific protein interactions during assembly of respiratory syncytial virus (RSV) are unknown. Here, the postulated interaction between the RSV matrix protein (M) and G glycoprotein (G) was investigated. Partial co-localization of M with G was demonstrated, but not with a truncated variant lacking the cytoplasmic domain and one-third of the transmembrane domain, in cells infected with recombinant RSV or transfected to express G and M. A series of G mutants was constructed with progressively truncated or modified cytoplasmic domains. Data from co-expression in cells and a cell-free binding assay showed that the N-terminal aa 2-6 of G play a key role in G-M interaction, with serine at position 2 and aspartate at position 6 playing key roles.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data from co-expression in cells and a cell-free binding assay showed that the N-terminal aa 2-6 of G play a key role in G-M interaction, with serine at position 2 and aspartate at position 6 playing key roles."}},"tag":"EXP"},{"id":115,"details":{"paperId":"4c3101ad804fdd846028d0e0837db5aacc7839d2","externalIds":{"MAG":"2021865874","DOI":"10.1016/S0168-1702(97)00121-4","CorpusId":"22039839","PubMed":"9617766"},"title":"Interactions between cellular actin and human respiratory syncytial virus (HRSV).","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"During HRSV infection in HEp-2 cells there are interactions between cellular actin and viral components, that can promote a transitory increase in the polymerization of synthetized actin, mainly of the beta isotype."}},"tag":"EXP"},{"id":307,"details":{"paperId":"038f0efccd3be95434cde7d2926fee0f88edfc4f","externalIds":{"MAG":"2153303340","DOI":"10.1128/JVI.01783-14","CorpusId":"36102341","PubMed":"25355874"},"title":"Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis","abstract":"ABSTRACT The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex, and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication."}},"tag":"EXP"},{"id":276,"details":{"paperId":"12973cf718343932aa2fe06d41fa3dfae3bb834b","externalIds":{"PubMedCentral":"7459546","MAG":"3039872072","DOI":"10.1128/JVI.00297-20","CorpusId":"220306257","PubMed":"32611756"},"title":"Interferon-Induced Protein 44 and Interferon-Induced Protein 44-Like Restrict Replication of Respiratory Syncytial Virus","abstract":"RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection. ABSTRACT Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection. IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A function for IFI44 and IFI 44L in controlling RSV infection is demonstrated, and it is hypothesized that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level."}},"tag":"EXP"},{"id":66,"details":{"paperId":"084c268da80ff2f5231df2e6f3569c4932d680a0","externalIds":{"PubMedCentral":"7317237","MAG":"3034677953","DOI":"10.1016/j.isci.2020.101256","CorpusId":"220060550","PubMed":"32580124"},"title":"Interleukin-22 Inhibits Respiratory Syncytial Virus Production by Blocking Virus-Mediated Subversion of Cellular Autophagy","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A previously unrecognized anti-viral effect of IL-22 that can be harnessed to prevent RSV-induced severe respiratory disease is informed."}},"tag":"EXP"},{"id":30,"details":{"paperId":"bc66c0cc08d90dbfbb634babcf385329eeeb181a","externalIds":{"MAG":"2081613292","DOI":"10.1007/BF01311117","CorpusId":"19990306","PubMed":"2719556"},"title":"Interprotein disulfide bonding between F and G glycoproteins of human respiratory syncytial virus","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results provide a direct evidence for the presence of envelope glycoprotein complexes linked by interprotein disulfide bonding, which may have implications on the structural and functional properties of envelope Glycoproteins."}},"tag":"EXP"},{"id":219,"details":{"paperId":"fc7067d79a36749d33986beab88475f3e8eac6e0","externalIds":{"MAG":"2012680472","DOI":"10.1099/0022-1317-73-5-1177","CorpusId":"25777610","PubMed":"1375280"},"title":"Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage.","abstract":"The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV. One of the recombinant viruses, vF317, expressed F protein (F317) that was processed like the RSV F glycoprotein. F317 was synthesized initially as F0, the uncleaved glycosylated precursor of mature F protein, and formed stable oligomeric structures that were maintained following cleavage of F0 to form the disulphide bond-linked F1 and F2 subunits. Most of the newly synthesized F0 expressed by either RSV or by vF317 was sensitive to treatment with endoglycosidase H (Endo H). Following cleavage of F0, F1 was resistant to Endo H, suggesting that conversion to complex-type sugars, which takes place in the medial Golgi apparatus, occurred simultaneously with or immediately prior to cleavage of F0 into F1 and F2. Another recombinant virus, vF313, synthesized only uncleaved F protein (F313) that comigrated with F0. Uncleaved F313 was expressed as a stable glycosylated protein; however, unlike cleaved F317, its oligosaccharides were not modified to complex forms, as determined from its Endo H sensitivity, and uncleaved F313 did not assemble into stable oligomeric structures. Nucleotide sequence analysis of the cDNA clones encoding F313 and F317 revealed four predicted amino acid sequence differences, none of which were located at the cleavage site. Expression of chimeric F proteins obtained by restriction fragment exchange between the two cDNA clones indicated that two amino acid changes in the F1 domain, located at amino acid residues 301 (Val to Ala) and 447 (Val to Met), resulted in the expression of uncleaved F protein. A change at either of these two amino acid residues, 301 or 447, resulted in the expression of inefficiently cleaved F protein, defining an additional F protein phenotype. Pulse-chase analyses to examine the association of recombinant F glycoproteins with gradient-purified fractionated membranes or with GRP78-BiP, a protein resident in the endoplasmic reticulum (ER) which binds to nascent proteins, revealed that uncleaved F protein (F313) is associated with GRP78-BiP in the ER for a longer time than F317, and little if any F313 was transported to the cell surface. In addition, the uncleaved F protein (F313) was not recognized by a panel of F protein-specific monoclonal antibodies in ELISA or indirect immunofluorescence assays, suggesting that F313 was misfolded and, as a result, not transported properly or cleaved.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The intracellular processing and transport of the respiratory syncytial virus (RSV) fusion (F) glycoprotein was examined by comparing the maturation and stability of wild-type F, uncleaved mutant F and chimeric F glycoproteins expressed by recombinant vaccinia viruses to that of F protein expressed by RSV."}},"tag":"EXP"},{"id":121,"details":{"paperId":"1b96918b4ddc92236ec80038a7ff265c1cd9a9e3","externalIds":{"MAG":"2290063637","DOI":"10.1021/acs.biochem.5b01332","CorpusId":"206514694","PubMed":"26901160"},"title":"Intrinsic Disorder to Order Transitions in the Scaffold Phosphoprotein P from the Respiratory Syncytial Virus RNA Polymerase Complex.","abstract":"Intrinsic disorder is at the center of biochemical regulation and is particularly overrepresented among the often multifunctional viral proteins. Replication and transcription of the respiratory syncytial virus (RSV) relies on a RNA polymerase complex with a phosphoprotein cofactor P as the structural scaffold, which consists of a four-helix bundle tetramerization domain flanked by two domains predicted to be intrinsically disordered. Because intrinsic disorder cannot be reduced to a defined atomic structure, we tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach. P remains as a tetramer above 70 °C but shows a pronounced reversible secondary structure transition between 10 and 60 °C. While the N-terminal module behaves as a random coil-like IDP in a manner independent of tetramerization, the isolated C-terminal module displays a cooperative and reversible metastable transition. When linked to the tetramerization domain, the C-terminal module becomes markedly more structured and stable, with strong ANS binding. Therefore, the tertiary structure in the C-terminal module is not compact, conferring \"late\" molten globule-like IDP properties, stabilized by interactions favored by tetramerization. The presence of a folded structure highly sensitive to temperature, reversibly and almost instantly formed and broken, suggests a temperature sensing activity. The marginal stability allows for exposure of protein binding sites, offering a thermodynamic and kinetic fine-tuning in order-disorder transitions, essential for the assembly and function of the RSV RNA polymerase complex.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This work tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach, finding the tertiary structure in the C-terminal module is not compact, conferring \"late\" molten globule-like IDP properties, stabilized by interactions favored by tetramerization."}},"tag":"EXP"},{"id":492,"details":{"paperId":"a1b7bcbed03e755e8ed6fdb6403f673b73748722","externalIds":{"PubMedCentral":"9820559","DOI":"10.3390/ijms24010569","CorpusId":"255287986","PubMed":"36614009"},"title":"Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization","abstract":"The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus."}},"tag":"EXP"},{"id":113,"details":{"paperId":"35d6c3ebfa295964aeeb2eea65604a8725c5a59c","externalIds":{"MAG":"2075995301","DOI":"10.1016/S0042-6822(02)00063-6","CorpusId":"34076026","PubMed":"12667822"},"title":"Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly."}},"tag":"EXP"},{"id":3,"details":{"paperId":"3ab77e32e2b5847864e46c0af9d386895e6ad79a","externalIds":{"MAG":"2118390380","DOI":"10.1002/jlb.66.1.50","CorpusId":"21113144","PubMed":"10410989"},"title":"Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells","abstract":"The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin‐dependent uptake mechanisms, endocytic pathways involving clathrin‐coated pits, and the mannose receptor. Furthermore, co‐localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established. J. Leukoc. Biol. 66: 50–58; 1999.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendrite cells to those established, adding to the list of routes established by respiratory syncytial virus infection."}},"tag":"EXP"},{"id":118,"details":{"paperId":"b2eff0dee109f141625d645d465db0cdf93cc421","externalIds":{"MAG":"2081189029","DOI":"10.1016/S1286-4579(02)00079-5","CorpusId":"19725490","PubMed":"12650770"},"title":"Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Findings indicate that inhibitors of annexin II could have potential in treating RSV infection, as isolated as a potential RSV receptor on Hep2 cells."}},"tag":"EXP"},{"id":455,"details":{"paperId":"16189f7ecaabd54ca1dce5f3da7c2af7521d79d6","externalIds":{"PubMedCentral":"6394897","MAG":"2917025683","DOI":"10.1371/journal.ppat.1007548","CorpusId":"71715471","PubMed":"30817806"},"title":"Killing two birds with one stone: How the respiratory syncytial virus polymerase initiates transcription and replication","abstract":"Respiratory syncytial virus (RSV) is the major cause of respiratory disease in infants and young children; it is also a significant problem in the elderly [1, 2]. RSV is a nonsegmented, negative strand RNA virus (nsNSV). Like other viruses in this group, the genome is a template for two distinct processes: transcription, which yields capped and polyadenylated mRNAs, and replication, which yields an encapsidated antigenome RNA. The antigenome, in turn, acts as a template for genome synthesis (Fig 1A; [3]). RSV encodes its own RNA-dependent RNA polymerase, which is responsible for both transcription and replication. This presents an intriguing puzzle, namely, how does the RSV polymerase perform both activities from the same template?","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV encodes its own RNA-dependent RNA polymerase, which is responsible for both transcription and replication, and this presents an intriguing puzzle, namely, how does the RSV polymerase perform both activities from the same template."}},"tag":"EXP"},{"id":213,"details":{"paperId":"8edfff52c6d237849266ba63a0c8406ce86e10cc","externalIds":{"MAG":"2028380385","DOI":"10.1099/0022-1317-69-2-313","CorpusId":"28830337","PubMed":"3339328"},"title":"Kinetics of synthesis and phosphorylation of respiratory syncytial virus polypeptides.","abstract":"The kinetics of synthesis of [35S]methionine-labelled respiratory syncytial virus-specific proteins were studied in CV-1 cells infected at high multiplicity. Immunoprecipitated viral proteins resolved by SDS-PAGE were quantified by scanning fluorographs of protein bands. The nucleocapsid (N) protein was detectable by 2 h post-infection (p.i.), whereas the phospho- (P), matrix (M) and fusion (Fo) proteins and Vp24 (a matrix-like protein) were first detected between 4 and 6 h p.i. Synthesis of the glyco- (G) protein was first detected at 6 h p.i. and reached its peak synthesis rate at 10 h p.i. Virus-specific P, M and Vp24 proteins were phosphorylated in infected cells. The P protein was highly phosphorylated in purified virions whereas phosphorylated species of the M and Vp24 proteins were minor components. The phosphorylated form of the P protein was detected by monoclonal antibody precipitation, confirming the identity of this protein. The N protein was not phosphorylated in infected cells or in virions. Synthesis of [35S]methionine-labelled proteins preceded detectable 32Pi labelling by several hours. The putative phosphorylated M protein was detected at 6 h p.i. before phosphorylated forms of P and Vp24 were seen. The timing of appearance of the phosphorylated species of P and Vp24 proteins in infected cells corresponded to the release of infectious virions from infected cell monolayers at 10 to 12 h p.i.","publicationTypes":["JournalArticle","Study"],"tldr":{"model":"tldr@v2.0.0","text":"The kinetics of synthesis of [35S]methionine-labelled respiratory syncytial virus-specific proteins were studied in CV-1 cells infected at high multiplicity and Virus-specific P, M and Vp24 proteins were phosphorylated in infected cells."}},"tag":"EXP"},{"id":2,"details":{"paperId":"afddd2e5e68c84e0daf4ad64be11cac4ce602bde","externalIds":{"MAG":"1966282529","DOI":"10.1002/eji.200324218","CorpusId":"22756073","PubMed":"14515273"},"title":"Lactoferrin and surfactant protein A exhibit distinct binding specificity to F protein and differently modulate respiratory syncytial virus infection","abstract":"Surfactant protein A (SP‐A) and lactoferrin (LF) play important roles in innate immune systems in the respiratory mucous membranes. We investigated how SP‐A and LF act against respiratory syncytial virus (RSV) infection. The present study indicated that RSV‐induced IL‐8 secretion from HEp‐2 cells was up‐regulated by SP‐A (170% of control) but down‐regulated by LF (23% of control). RSV infectivity determined by viral titers and the uptake of FITC‐labeled RSV were also increased by SP‐A, but decreased by LF. To clarify the mechanism of these opposite effects, we examined the interactions of SP‐A and LF with RSV F protein, the most important surface glycoprotein for viral penetration. RSV F protein was found to be the ligand for both SP‐A and LF, but the manners of binding were different. LF directly interacted with the F1 subunit, which involved antigenic sites of F protein. Contrarily, SP‐A associated with the F2 subunit, which was highly glycosylated. SP‐A but not LF failed to interact with deglycosylated F protein. Moreover, SP‐A initiated the hemolyzing fusion activity of F protein. These results suggest that SP‐A and LF modulate RSV infection by different binding specificity to F protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that SP‐A and LF modulate RSV infection by different binding specificity to F’protein, the most important surface glycoprotein for viral penetration."}},"tag":"EXP"},{"id":262,"details":{"paperId":"00998c7c5e7456031e833dc047c28e859bf3a75f","externalIds":{"MAG":"2890780049","DOI":"10.1111/cmi.12955","CorpusId":"52291383","PubMed":"30223301"},"title":"Late activation of the Raf/MEK/ERK pathway is required for translocation of the respiratory syncytial virus F protein to the plasma membrane and efficient viral replication","abstract":"Activation of the Raf/MEK/ERK cascade is required for efficient propagation of several RNA and DNA viruses, including human respiratory syncytial virus (RSV). In RSV infection, activation of the Raf/MEK/ERK cascade is biphasic. An early induction within minutes after infection is associated with viral attachment. Subsequently, a second activation occurs with, so far, unknown function in the viral life cycle. In this study, we aimed to characterise the role of Raf/MEK/ERK‐mediated signalling during ongoing RSV infection. Our data show that inhibition of the kinase MEK after the virus has been internalised results in a reduction of viral titers. Further functional investigations revealed that the late‐stage activation of ERK is required for a specific step in RSV replication, namely, the secretory transport of the RSV fusion protein F. Thus, MEK inhibition resulted in impaired surface accumulation of the F protein. F protein surface expression is essential for efficient replication as it is involved in viral filament formation, cell fusion, and viral transmission. In summary, we provide detailed insights of how host cell signalling interferes with RSV replication and identified the Raf/MEK/ERK kinase cascade as potential target for novel anti‐RSV strategies.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Detailed insights of how host cell signalling interferes with RSV replication are provided and the Raf/MEK/ERK kinase cascade is identified as potential target for novel anti‐RSV strategies."}},"tag":"EXP"},{"id":216,"details":{"paperId":"9f95390c6c64e61b87603713e579a91594cf79a2","externalIds":{"MAG":"2118280608","DOI":"10.1099/0022-1317-72-6-1455","CorpusId":"24154176","PubMed":"2045795"},"title":"Location of phosphorylated residues in human respiratory syncytial virus phosphoprotein.","abstract":"The phosphoprotein (P protein) from human respiratory syncytial virus Long strain, labelled in vivo with [32P]orthophosphate, was purified from virions or virus-infected human epithelial (Hep-2) cells. The main phosphorylated amino acid found was serine. The determination of the N-terminal sequence of unphosphorylated and phosphorylated fragments of P protein obtained after chemical or enzymic treatments suggested that some or all of the six serines present at positions 116, 117, 119, 143, 156 and 161 are the major phosphorylated residues, although a modification in serine residues at positions 86, 94 and 99 can not be ruled out.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The determination of the N-terminal sequence of unphosphorylated and phosphorylated fragments of P protein obtained after chemical or enzymic treatments suggested that some or all of the six serines present at positions 116, 117, 119, 143, 156 and 161 are the major phosphorylation residues, although a modification in serine residues can not be ruled out."}},"tag":"EXP"},{"id":271,"details":{"paperId":"d09c4bb769b40b84f9ab8dd92b71e1c35233d962","externalIds":{"PubMedCentral":"7199404","MAG":"3008450997","DOI":"10.1128/JVI.00113-20","CorpusId":"218685343"},"title":"Long Noncoding RNA NRAV Promotes Respiratory Syncytial Virus Replication by Targeting the MicroRNA miR-509-3p/Rab5c Axis To Regulate Vesicle Transportation","abstract":"The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection. ABSTRACT Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler’s (NeST) showed no difference in vivo and in vitro. Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus. IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"This study found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduced expression was part of the host antiviralresponse."}},"tag":"EXP"},{"id":192,"details":{"paperId":"25e667e7d825e68d0f01548edd4d7513e10ccf1e","externalIds":{"MAG":"2042975473","DOI":"10.1089/VIM.2000.13.125","CorpusId":"6228998","PubMed":"10733174"},"title":"Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells.","abstract":"Lung surfactant protein A (SP-A) has a central role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-A. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G-protein from RSV (human, A2 strain) and human SP-A. The effect of SP-A on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and by interleukin-8 (IL-8) release from buffy coat (BC) cells. SP-A binds to RSV G-protein in a concentration-dependent manner that is inhibitable by both ethylenediamine tetraacetic acid (EDTA) and mannan, indicating that binding is through the carbohydrate recognition domain of the SP-A and a carbohydrate moiety of the G-protein. The level of RSV infection of Hep-2C cells increases with increasing concentrations of SP-A. The amount of IL-8 released by BC cells in the presence of RSV is increased with SP-A concentrations of 2.9 microg/mL or greater. Our results show that SP-A enhances the attachment of RSV and subsequent entry into host cells. The effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"SP-A enhances the attachment of RSV and subsequent entry into host cells and the effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV."}},"tag":"EXP"},{"id":202,"details":{"paperId":"07e58b5e6cb28085081b73ac2751c1cda84babe8","externalIds":{"MAG":"2806655355","PubMedCentral":"6061868","DOI":"10.1093/nar/gky480","CorpusId":"46949770","PubMed":"29873775"},"title":"Mechanism for de novo initiation at two sites in the respiratory syncytial virus promoter","abstract":"Abstract The respiratory syncytial virus (RSV) RNA dependent RNA polymerase (RdRp) initiates two RNA synthesis processes from the viral promoter: genome replication from position 1U and mRNA transcription from position 3C. Here, we examined the mechanism by which a single promoter can direct initiation from two sites. We show that initiation at 1U and 3C occurred independently of each other, and that the same RdRp was capable of precisely selecting the two sites. The RdRp preferred to initiate at 3C, but initiation site selection could be modulated by the relative concentrations of ATP versus GTP. Analysis of template mutations indicated that the RdRp could bind ATP and CTP, or GTP, independently of template nucleotides. The data suggest a model in which innate affinity of the RdRp for particular NTPs, coupled with a repeating element within the promoter, allows precise initiation of replication at 1U or transcription at 3C.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest a model in which innate affinity of the RdRp for particular NTPs, coupled with a repeating element within the promoter, allows precise initiation of replication at 1U or transcription at 3C."}},"tag":"EXP"},{"id":221,"details":{"paperId":"5ae7aec648e064f112ef065fbebd07cc3d889b88","externalIds":{"MAG":"2104885633","DOI":"10.1099/0022-1317-74-7-1445","CorpusId":"2510857","PubMed":"8336126"},"title":"Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus.","abstract":"Previous work has demonstrated that the small hydrophobic (SH) protein of human respiratory syncytial virus (RSV) A2 strain is a 64 amino acid integral membrane protein that accumulates intracellularly as an unglycosylated major species (SH0), a minor species truncated at the amino terminus and two N-glycosylated species one of which contains a further addition of polylactosamine. In this study, the membrane orientation of SH0 was mapped by trypsinization of intact RSV-infected cells followed by washout, lysis and immunoprecipitation of protected fragments with antisera specific for the protein termini. This showed that the C terminus is extracellular and the SH protein was not detectably palmitylated. Analysis of the SH protein by sedimentation on sucrose gradients showed that it rapidly assembles into a homo-oligomer that co-sediments with the F protein tetramer. Interestingly, all forms of the SH protein were found in the oligomeric fraction. Chemical cross-linking generated species which appeared to represent dimers, trimers, tetramers and pentamers as well as a minor species of 180K which might correspond to the oligomeric form detected by sucrose gradient sedimentation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The membrane orientation of SH0 was mapped by trypsinization of intact RSV-infected cells followed by washout, lysis and immunoprecipitation of protected fragments with antisera specific for the protein termini showed that the C terminus is extracellular and the SH protein was not detectably palmitylated."}},"tag":"EXP"},{"id":13,"details":{"paperId":"2e609be176d30924591a68a3d318e2552753a330","externalIds":{"MAG":"1997699232","DOI":"10.1006/VIRO.1997.8696","CorpusId":"24180155","PubMed":"9281514"},"title":"Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus.","abstract":"The small hydrophobic (SH) protein of human respiratory syncytial virus (HRSV) has been efficiently expressed in Escherichia coli. In analogy to small hydrophobic proteins encoded by other RNA viruses, membrane permeability changes to low-molecular-weight compounds were detected in bacteria expressing HRSV SH protein. These changes implied, at least, the entry of both the protein synthesis inhibitor hygromycin B and the beta-galactoside substrate o-nitrophenyl-beta-d-galactopyranoside and the exit of preloaded [3H]uridine from bacterial cells. Site-directed mutagenesis indicated that the C-terminal end of SH is needed for induction of membrane permeability changes. In addition, amino acid substitution at residue 32 (Ile to Lys) abolished that activity. This was correlated with a drastic increase in SH electrophoretic mobility and a decrease of the predicted values of alpha-helix for all residues of the SH transmembrane domain. Other sequence changes have either partial effect or no effect on the membrane permeability changes induced by the SH protein. However, none of the mutations abrogated the association of SH protein with bacterial membranes, indicating that incorporation of SH protein to membranes is not sufficient to induce the observed changes. Membrane permeability changes then might provide a useful test for the identification of key amino acid residues in this unique HRSV gene product.","publicationTypes":["JournalArticle"],"tldr":null},"tag":"EXP"},{"id":397,"details":{"paperId":"910bed8d05fe133fca1da835a72a1ca79d5ed341","externalIds":{"MAG":"3087781123","PubMedCentral":"7512546","DOI":"10.1128/mBio.01202-20","CorpusId":"221861842","PubMed":"32963000"},"title":"Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro","abstract":"Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions. ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The domains of P required for IB formation are identified and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis, which strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics."}},"tag":"EXP"},{"id":58,"details":{"paperId":"92f1a13e28777f0b7706c10b869ebb300c71d029","externalIds":{"MAG":"2618093788","DOI":"10.1016/j.bbrc.2017.05.171","CorpusId":"205952485","PubMed":"28576489"},"title":"Mitochondrial protein p32/HAPB1/gC1qR/C1qbp is required for efficient respiratory syncytial virus production.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV replication as well as infectious virus production was significantly reduced by p32 siRNA knockdown, consistent with an important role for p32 in RSV infection."}},"tag":"EXP"},{"id":356,"details":{"paperId":"cd12e988d2e79f0b538d04b5b0eaedbf0d070a45","externalIds":{"MAG":"2158773508","DOI":"10.1128/JVI.73.1.388-397.1999","CorpusId":"26868525","PubMed":"9847343"},"title":"Model for Polymerase Access to the Overlapped L Gene of Respiratory Syncytial Virus","abstract":"ABSTRACT The last two genes of respiratory syncytial virus (RSV), M2 and L, overlap by 68 nucleotides, an arrangement which has counterparts in a number of nonsegmented negative-strand RNA viruses. Thus, the gene-end (GE) signal of M2 lies downstream of the L gene-start (GS) signal, separated by 45 nucleotides. Since RSV transcription ostensibly is sequential and unidirectional from a single promoter within the 3′ leader region, it was unclear how the polymerase accesses the L GS signal. Furthermore, it was previously shown that 90% of transcripts which are initiated at the L GS signal are polyadenylated and terminated at the M2 GE signal, yielding a short, truncated L mRNA as the major transcription product of the L gene. Despite these apparent down-regulatory features, we show that the accumulation of full-length L mRNA during RSV infection is only sixfold less than that of its upstream neighbor, M2. We used cDNA-encoded genome analogs in an intracellular transcription assay to investigate the mechanism of transcription of the overlapped genes. Expression of L was found to be dependent on sequential transcription from the 3′ end of the genome. Apart from the L GS signal, the only other strict requirement for initiation at L was the M2 GE signal. This implies that the polymerase accesses the L GS signal only following arrival at the M2 GE signal. Thus, polymerase which terminates at the M2 GE signal presumably scans upstream to initiate at the L GS signal. This also would provide a mechanism whereby polymerase which terminates prematurely during transcription of L could recycle from the M2 GE signal to the L GS signal, thereby accounting for the unexpectedly high level of synthesis of full-length L mRNA. The sequence and spacing between the two signals were not critical. Furthermore, the polymerase also was capable of efficiently transcribing from an L GS signal placed downstream of the M2 GE signal, implying that the overlapping arrangement is not obligatory. When copies of the L GS signal were placed concurrently upstream and downstream of the M2 GE signal, both were utilized. This finding indicates that a polymerase situated at a GE signal is capable of scanning for a GS signal in either the upstream or downstream direction and thereafter initiating transcription.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This finding indicates that a polymerase situated at a GE signal is capable of scanning for a GS signal in either the upstream or downstream direction and thereafter initiating transcription, thereby accounting for the unexpectedly high level of synthesis of full-length L mRNA."}},"tag":"EXP"},{"id":127,"details":{"paperId":"ef9a18949db179d4b805842ea56c2d2dca82578e","externalIds":{"MAG":"2067461300","DOI":"10.1021/bi300765c","CorpusId":"28848736","PubMed":"22978633"},"title":"Modular unfolding and dissociation of the human respiratory syncytial virus phosphoprotein p and its interaction with the m(2-1) antiterminator: a singular tetramer-tetramer interface arrangement.","abstract":"Paramyxoviruses share the essential RNA polymerase complex components, namely, the polymerase (L), phosphoprotein (P), and nucleoprotein (N). Human respiratory syncytial virus (RSV) P is the smallest polypeptide among the family, sharing a coiled coil tetramerization domain, which disruption renders the virus inactive. We show that unfolding of P displays a first transition with low cooperativity but substantial loss of α-helix content and accessibility to hydrophobic sites, indicative of loose chain packing and fluctuating tertiary structure, typical of molten globules. The lack of unfolding baseline indicates a native state in conformational exchange and metastable at 20 °C. The second transition starts from a true intermediate state, with only the tetramerization domain remaining folded. The tetramerization domain undergoes a two-state dissociation/unfolding reaction (37.3 kcal mol(-1)). The M(2-1) transcription antiterminator, unique to RSV and Metapneumovirus, forms a nonglobular P:M(2-1) complex with a 1:1 stoichiometry and a K(D) of 8.1 nM determined by fluorescence anisotropy, far from the strikingly coincident dissociation range of P and M(2-1) tetramers (10(-28) M(3)). The M(2-1) binding region has been previously mapped to the N-terminal module of P, strongly suggesting the latter as the metastable molten globule domain. Folding, oligomerization, and assembly events between proteins and with RNA are coupled in the RNA polymerase complex. Quantitative assessment of the hierarchy of these interactions and their mechanisms contribute to the general understanding of RNA replication and transcription in Paramyxoviruses. In particular, the unique P-M(2-1) interface present in RSV provides a valuable antiviral target for this worldwide spread human pathogen.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that unfolding of P displays a first transition with low cooperativity but substantial loss of α-helix content and accessibility to hydrophobic sites, indicative of loose chain packing and fluctuating tertiary structure, typical of molten globules."}},"tag":"EXP"},{"id":510,"details":{"paperId":"e639408c75bc68ecc48bc314bec3c3bebb849f4b","externalIds":{"MAG":"1976420475","PubMedCentral":"4189880","DOI":"10.4161/viru.32225","CorpusId":"639104","PubMed":"25513775"},"title":"Modulation of host adaptive immunity by hRSV proteins","abstract":"Globally, the human respiratory syncytial virus (hRSV) is the major cause of lower respiratory tract infections (LRTIs) in infants and children younger than 2 years old. Furthermore, the number of hospitalizations due to LRTIs has shown a sustained increase every year due to the lack of effective vaccines against hRSV. Thus, this virus remains as a major public health and economic burden worldwide. The lung pathology developed in hRSV-infected humans is characterized by an exacerbated inflammatory and Th2 immune response. In order to rationally design new vaccines and therapies against this virus, several studies have focused in elucidating the interactions between hRSV virulence factors and the host immune system. Here, we discuss the main features of hRSV biology, the processes involved in virus recognition by the immune system and the most relevant mechanisms used by this pathogen to avoid the antiviral host response.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The main features of hRSV biology, the processes involved in virus recognition by the immune system and the most relevant mechanisms used by this pathogen to avoid the antiviral host response are discussed."}},"tag":"EXP"},{"id":495,"details":{"paperId":"adb408713c36d66581b6ff4bb41b199e01697f9d","externalIds":{"MAG":"2793420120","PubMedCentral":"5869502","DOI":"10.3390/v10030109","CorpusId":"3729292","PubMed":"29510513"},"title":"Molecular Requirements for Self-Interaction of the Respiratory Syncytial Virus Matrix Protein in Living Mammalian Cells","abstract":"Respiratory syncytial virus (RSV) is an important human pathogen, which infects respiratory tract epithelial cells causing bronchiolitis and pneumonia in children and the elderly. Recent studies have linked RSV matrix (M) ability to self-interaction and viral budding. However, RSV M has been crystalized both as a monomer and a dimer, and no formal proof exists to date that it forms dimers in cells. Here, by using a combination of confocal laser scanning microscopy and bioluminescent resonant energy transfer applied to differently tagged deletion mutants of RSV M, we show that the protein can self-interact in living mammalian cells and that both the N and C-terminus of the protein are strictly required for the process, consistent with the reported dimeric crystal structure.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that the RSV matrix protein can self-interact in living mammalian cells and that both the N and C-terminus of the protein are strictly required for the process, consistent with the reported dimeric crystal structure."}},"tag":"EXP"},{"id":474,"details":{"paperId":"d0604339593062b768b27a7cdf52251c1fc6787a","externalIds":{"MAG":"2132360000","DOI":"10.2217/fmb.12.132","CorpusId":"19059213","PubMed":"23252497"},"title":"Molecular mechanisms driving respiratory syncytial virus assembly.","abstract":"Respiratory syncytial virus is a single-stranded RNA virus in the Paramyxoviridae family that preferentially assembles and buds from the apical surface of polarized epithelial cells, forming filamentous structures that contain both viral proteins and the genomic RNA. Recent studies have described both viral and host factors that are involved in ribonucleoprotein assembly and trafficking of viral proteins to the cell surface. At the cell surface, viral proteins assemble into filaments that probably require interactions between viral proteins, host proteins and the cell membrane. Finally, a membrane scission event must occur to release the free virion. This article will review the recent literature describing the mechanisms that drive respiratory syncytial virus assembly and budding.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This article will review the recent literature describing the mechanisms that drive respiratory syncytial virus assembly and budding."}},"tag":"EXP"},{"id":280,"details":{"paperId":"938961f2fadb7bba0c50afbad3ecc69cfac24937","externalIds":{"MAG":"2096886486","DOI":"10.1128/JVI.00413-11","CorpusId":"206793798","PubMed":"21795342"},"title":"Multiple Functional Domains and Complexes of the Two Nonstructural Proteins of Human Respiratory Syncytial Virus Contribute to Interferon Suppression and Cellular Location","abstract":"ABSTRACT Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKKε, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKKε reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo- as well as heteromers, and it is suggested that the NS1-NS2 heteromer localizes to the mitochondria."}},"tag":"EXP"},{"id":229,"details":{"paperId":"4cc76e97f4cc8e15c0a90ab7370810162bd7edce","externalIds":{"MAG":"1682638332","DOI":"10.1099/0022-1317-83-1-61","CorpusId":"5609254","PubMed":"11752701"},"title":"Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells.","abstract":"Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2](a/b). Enzymatic deglycosylation of [F2](a/b) produced a single 10 kDa product suggesting that [F2](a/b) arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2](a/b) was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2](a/b), via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a and F2b, which were collectively referred to as [F2](a/b)."}},"tag":"EXP"},{"id":33,"details":{"paperId":"772634b556d225978ac2a35917479a40fae5cd87","externalIds":{"MAG":"2025510141","DOI":"10.1007/s00705-003-0139-0","CorpusId":"11815871","PubMed":"14551820"},"title":"Multiple heparin binding domains of respiratory syncytial virus G mediate binding to mammalian cells","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data show that the G HBD includes as much as residues 187–231, that there is redundancy beyond the previously described HBD, and that the cell-binding and virus infectivity-blocking functions of these recombinant G proteins were closely linked and required at least one HBD."}},"tag":"EXP"},{"id":369,"details":{"paperId":"92ac77ea65c0c84104d9d409dceed6aae06ea09c","externalIds":{"MAG":"2051845598","DOI":"10.1128/JVI.75.10.4744-4751.2001","CorpusId":"10723215","PubMed":"11312346"},"title":"N-Glycans of F Protein Differentially Affect Fusion Activity of Human Respiratory Syncytial Virus","abstract":"ABSTRACT The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F1 subunit attached to N500, however, is required for efficient syncytium formation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the P protein, while a triple mutant devoid of any N-linked oligosaccharide was generated."}},"tag":"EXP"},{"id":184,"details":{"paperId":"94a46df36753299251ede6fdf9d93811b1c34857","externalIds":{"MAG":"2124107211","DOI":"10.1074/mcp.M114.044107","CorpusId":"25683946","PubMed":"25556234"},"title":"New Host Factors Important for Respiratory Syncytial Virus (RSV) Replication Revealed by a Novel Microfluidics Screen for Interactors of Matrix (M) Protein*","abstract":"Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking, which have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell."}},"tag":"EXP"},{"id":176,"details":{"paperId":"de3dc5ed757b139bbe60eeb4141d2c6824ae3d08","externalIds":{"MAG":"2562682816","DOI":"10.1074/jbc.M116.765958","CorpusId":"205363034","PubMed":"28031463"},"title":"New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners*","abstract":"Phosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales. It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that N- and C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N-terminal region. They all mediate internal long-range contacts in this non-globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown here that N- and C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus."}},"tag":"EXP"},{"id":476,"details":{"paperId":"271ea603af63bea084f7b26780787acd50e8569d","externalIds":{"MAG":"1671409694","DOI":"10.3109/1040841X.2015.1055711","CorpusId":"26111609","PubMed":"26119025"},"title":"New insights on the viral and host factors contributing to the airway pathogenesis caused by the respiratory syncytial virus","abstract":"Abstract The respiratory syncytial virus (RSV) is the most prevalent etiological agent of lower respiratory tract infections and the first cause of hospitalization in infants due to respiratory disease worldwide. However, efforts to develop safe and effective vaccines and antivirals have been challenged by an incomplete understanding of the RSV pathogenesis and the host immune response to RSV infection in the airways. Here, we discuss recent advances in understanding the interaction between RSV and the epithelium to induce pathogenesis in the airways, such as the role of the RSV NS2 protein in the airway epithelium, as well as the events involved in the RSV entry process. In addition, we summarize the cellular factors produced by airway epithelial cells (AECs) in response to RSV infection that lead to the activation of innate and adaptive immune responses, inducing lung inflammation and disease. Further, we discuss the possible contribution of a recently identified cytokine, thymic stromal lymphopoitein (TSLP), in the lung immunopathology caused by RSV.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Recent advances in understanding the interaction between RSV and the epithelium to induce pathogenesis in the airways are discussed, such as the role of the RSV NS2 protein in theAirway epithelial cells, as well as the events involved in theRSV entry process."}},"tag":"EXP"},{"id":395,"details":{"paperId":"c8d24e8fa8a94e72aafc3107c99da07066f1d3de","externalIds":{"MAG":"2944326410","PubMedCentral":"6509182","DOI":"10.1128/mBio.00187-19","CorpusId":"147706533","PubMed":"31064823"},"title":"No Incongruity in Respiratory Syncytial Virus M2-1 Protein Remaining Bound to Viral mRNAs during Their Entire Life Time","abstract":"Selvaraj et al. recently published the structure of the M2-1 protein of respiratory syncytial virus (RSV) bound to its interaction domain on the P protein (1). These results constitute an important step toward the understanding of RSV transcription process and, on a broader level, of RSV RNA-dependent RNA polymerase regulation and functioning. Interestingly, their results suggest that M2-1 could play a posttranscriptional role in viral mRNA metabolism which is consistent with …","publicationTypes":["LettersAndComments"],"tldr":{"model":"tldr@v2.0.0","text":"Interestingly, their results suggest that M2-1 could play a posttranscriptional role in viral mRNA metabolism which is consistent with that in RSV transcription process."}},"tag":"EXP"},{"id":119,"details":{"paperId":"b3d39a31e5fe5f518c44d4d2831830871bd5f4f4","externalIds":{"MAG":"2889502637","DOI":"10.1021/acs.analchem.8b02402","CorpusId":"52100998","PubMed":"30152690"},"title":"Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein.","abstract":"Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The discovery of a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety."}},"tag":"EXP"},{"id":480,"details":{"paperId":"a40ce33a08c45025677374a4c841ba71f7d82aea","externalIds":{"PubMedCentral":"6292865","MAG":"2903225001","DOI":"10.3389/fimmu.2018.02872","CorpusId":"54447224","PubMed":"30581436"},"title":"Non-specific Effects of Live Attenuated Pertussis Vaccine Against Heterologous Infectious and Inflammatory Diseases","abstract":"Bordetella pertussis is the agent of pertussis, also referred to as whooping cough, a disease that remains an important public health issue. Vaccine-induced immunity to pertussis wanes over time. In industrialized countries, high vaccine coverage has not prevented infection and transmission of B. pertussis, leading to periodic outbreaks in people of all ages. The consequence is the formation of a large source for transmission to children, who show the highest susceptibility of developing severe whooping cough and mortality. With the aim of providing protection against both disease and infection, a live attenuated pertussis vaccine, in which three toxins have been genetically inactivated or removed, is now in clinical development. This vaccine, named BPZE1, offers strong protection in mice and non-human primates. It has completed a phase I clinical trial in which safety, transient colonization of the human airway and immunogenicity could be demonstrated. In mice, BPZE1 was also found to protect against inflammation resulting from heterologous airway infections, including those caused by other Bordetella species, influenza virus and respiratory syncytial virus. Furthermore, the heterologous protection conferred by BPZE1 was also observed for non-infectious inflammatory diseases, such as allergic asthma, as well as for inflammatory disorders outside of the respiratory tract, such as contact dermatitis. Current studies focus on the mechanisms underlying the anti-inflammatory effects associated with nasal BPZE1 administration. Given the increasing importance of inflammatory disorders, novel preventive and therapeutic approaches are urgently needed. Therefore, live vaccines, such as BPZE1, may offer attractive solutions. It is now essential to understand the cellular and molecular mechanisms of action before translating these biological findings into new healthcare solutions.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In mice, BPZE1 was also found to protect against inflammation resulting from heterologous airway infections, including those caused by other Bordetella species, influenza virus and respiratory syncytial virus, as well as for inflammatory disorders outside of the respiratory tract, such as contact dermatitis."}},"tag":"EXP"},{"id":287,"details":{"paperId":"cad1305d750d97262d13dc539cf91fb9b18b79ac","externalIds":{"MAG":"2062215675","DOI":"10.1128/JVI.00630-08","CorpusId":"9366902","PubMed":"18562519"},"title":"Nonstructural Proteins 1 and 2 of Respiratory Syncytial Virus Suppress Maturation of Human Dendritic Cells","abstract":"ABSTRACT Human respiratory syncytial virus (RSV) is the most important agent of serious pediatric respiratory tract disease worldwide. One of the main characteristics of RSV is that it readily reinfects and causes disease throughout life without the need for significant antigenic change. The virus encodes nonstructural protein 1 (NS1) and NS2, which are known to suppress type I interferon (IFN) production and signaling. In the present study, we monitored the maturation of human monocyte-derived myeloid dendritic cells (DC) following inoculation with recombinant RSVs bearing deletions of the NS1 and/or NS2 proteins and expressing enhanced green fluorescent protein. Deletion of the NS1 protein resulted in increased expression of cell surface markers of DC maturation and an increase in the expression of multiple cytokines and chemokines. This effect was enhanced somewhat by further deletion of the NS2 protein, although deletion of NS2 alone did not have a significant effect. The upregulation was largely inhibited by pretreatment with a blocking antibody against the type I IFN receptor, suggesting that suppression of DC maturation by NS1/2 is, at least in part, a result of IFN antagonism mediated by these proteins. Therefore, this study identified another effect of the NS1 and NS2 proteins. The observed suppression of DC maturation may result in decreased antigen presentation and T-lymphocyte activation, leading to incomplete and/or weak immune responses that might contribute to RSV reinfection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The observed suppression of DC maturation by NS1/2 is, at least in part, a result of IFN antagonism mediated by these proteins, which may result in decreased antigen presentation and T-lymphocyte activation, leading to incomplete and/or weak immune responses that might contribute to RSV reinfection."}},"tag":"EXP"},{"id":301,"details":{"paperId":"e01a12e507ed239ec50dc1614a43db7c53638e11","externalIds":{"MAG":"2132937793","DOI":"10.1128/JVI.01420-06","CorpusId":"25359815","PubMed":"17151097"},"title":"Nonstructural Proteins of Respiratory Syncytial Virus Suppress Premature Apoptosis by an NF-κB-Dependent, Interferon-Independent Mechanism and Facilitate Virus Growth","abstract":"ABSTRACT The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-κB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is concluded that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway."}},"tag":"EXP"},{"id":412,"details":{"paperId":"3a8082b6b9aa267d96c01964236caf9ca42eb8ab","externalIds":{"MAG":"2066748639","DOI":"10.1159/000196385","CorpusId":"46834446","PubMed":"7716351"},"title":"Nonstructural protein 2 (NS2) of respiratory syncytial virus (RSV) detected by an antipeptide serum.","abstract":"The human respiratory syncytial virus (RSV) is often associated with airway obstruction and is suspected to induce bronchial hyperreactivity. Interactions of viral proteins with cellular components may be responsible for epithelial damage leading to bronchial hyperreactivity. In this study, we describe the localization of the 14.7-kD nonstructural protein 2 (NS2) in RSV-infected cells. The detection of NS2 was performed using antipeptide antibodies elicited against amino acids 109-123 of the predicted sequence of the NS2 protein. By using recombinant NS2, we could clearly demonstrate the specificity of the antipeptide antibodies. With this defined tool, NS2 could be first detected in infected HEp-2 cells at 10 h p.i. subsequently to the detection of N protein. In double-staining experiments, colocalization of NS2, P protein and N protein was demonstrated. The antipeptide antibodies recognized the NS2 protein in the sediment of RSV-infected HEp-2 cells lysed with RIPA buffer at 48 h p.i. The results agree with the reported interaction of RSV with cytoskeletal intermediate filaments. These interactions may implicate essential cellular functions suspected to induce bronchial hyperreactivity.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The localization of the 14.7-kD nonstructural protein 2 (NS2) in RSV-infected cells is described and these interactions may implicate essential cellular functions suspected to induce bronchial hyperreactivity."}},"tag":"EXP"},{"id":40,"details":{"paperId":"72d7e98a0a9efc037544836b8b362551ebbffc29","externalIds":{"DOI":"10.1007/s11033-012-2107-9","CorpusId":"254840819"},"title":"Nonstructural protein-1 of respiratory syncytial virus regulates HOX gene expression through interacting with histone","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is indicated that RSV NS-1 induces HOX gene expression, through histone ubiquitination in a BEC cell line, which may provide a novel conception for understanding the relationship between severe RSV bronchiolitis during early life and the development of subsequent asthma."}},"tag":"EXP"},{"id":469,"details":{"paperId":"9602f95b6a54d07adc1703ef06a02ec2e8a9689c","externalIds":{"MAG":"2234575765","PubMedCentral":"4819838","DOI":"10.1586/14787210.2016.1141676","CorpusId":"18309202","PubMed":"26760927"},"title":"Novel insights into human respiratory syncytial virus-host factor interactions through integrated proteomics and transcriptomics analysis","abstract":"ABSTRACT The lack of vaccine and limited antiviral options against respiratory syncytial virus (RSV) highlights the need for novel therapeutic strategies. One alternative is to develop drugs that target host factors required for viral replication. Several microarray and proteomics studies had been published to identify possible host factors that are affected during RSV replication. In order to obtain a comprehensive understanding of RSV-host interaction, we integrated available proteome and transcriptome datasets and used it to construct a virus-host interaction network. Then, we interrogated the network to identify host factors that are targeted by the virus and we searched for drugs from the DrugBank database that interact with these host factors, which may have potential applications in repositioning for future treatment options of RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This work integrated available proteome and transcriptome datasets and used it to construct a virus-host interaction network and searched for drugs from the DrugBank database that interact with these host factors that may have potential applications in repositioning for future treatment options of RSV infection."}},"tag":"EXP"},{"id":491,"details":{"paperId":"4e69146dfb69cb9fb949d0e1ea02c16403e8782c","externalIds":{"PubMedCentral":"9317576","DOI":"10.3390/ijms23147976","CorpusId":"250937177","PubMed":"35887322"},"title":"Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites","abstract":"Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells."}},"tag":"EXP"},{"id":125,"details":{"paperId":"9831acb22b657bd975c719a28f438d71a6177358","externalIds":{"MAG":"2014812468","DOI":"10.1021/BI050701E","CorpusId":"22081037","PubMed":"16171404"},"title":"Nuclear import of the respiratory syncytial virus matrix protein is mediated by importin beta1 independent of importin alpha.","abstract":"The matrix (M) protein of respiratory syncytial virus (RSV) plays an important role in virus assembly through specific interactions with RSV nucleocapsids and envelope glycoproteins in the cytoplasm as well as with the host cell membrane. We have previously shown that M localizes to the nucleus of infected cells at an early stage in the RSV infection cycle, where it may be instrumental in inhibiting host cell processes. The present study uses transient expression of M as well as a truncated green fluorescent protein (GFP) fusion derivative to show for the first time that M is able to localize in the nucleus in the absence of other RSV gene products, through the action of amino acids 110-183, encompassing the nucleic acid binding regions of the protein, that are sufficient to target GFP to the nucleus. Using native PAGE, ELISA-based binding assays, a novel Alphascreen assay, and an in vitro nuclear transport assay, we show that M is recognized directly by the importin beta1 nuclear import receptor, which mediates its nuclear import in concert with the guanine nucleotide-binding protein Ran. Retention of M in the nucleus through binding to nuclear components, probably mediated by the putative zinc finger domain of M, also contributes to M nuclear accumulation. This is the first report of the importin binding and nuclear import properties of a gene product from a negative sense RNA virus, with implications for the function of RSV M and possibly other viral M proteins in the nucleus of infected cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This is the first report of the importin binding and nuclear import properties of a gene product from a negative sense RNA virus, with implications for the function of RSV M and possibly other viral M proteins in the nucleus of infected cells."}},"tag":"EXP"},{"id":60,"details":{"paperId":"378837aabdb011ee27587a252db7524481e853af","externalIds":{"PubMedCentral":"8609347","DOI":"10.1016/j.celrep.2021.109803","CorpusId":"238858755","PubMed":"34644581"},"title":"Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription."}},"tag":"EXP"},{"id":513,"details":{"paperId":"0be6ab7e7b78081ef3fed60d5fc4ad85fd5c3d7e","externalIds":{"MAG":"2289237903","PubMedCentral":"4798948","DOI":"10.7554/eLife.12627","CorpusId":"1929954","PubMed":"26880565"},"title":"Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein","abstract":"Non-segmented, (-)RNA viruses cause serious human diseases. Human metapneumovirus (HMPV), an emerging pathogen of this order of viruses (Mononegavirales) is one of the main causes of respiratory tract illness in children. To help elucidate the assembly mechanism of the nucleocapsid (the viral RNA genome packaged by the nucleoprotein N) we present crystallographic structures of HMPV N in its assembled RNA-bound state and in a monomeric state, bound to the polymerase cofactor P. Our structures reveal molecular details of how P inhibits the self-assembly of N and how N transitions between the RNA-free and RNA-bound conformational state. Notably, we observe a role for the C-terminal extension of N in directly preventing premature uptake of RNA by folding into the RNA-binding cleft. Our structures suggest a common mechanism of how the growth of the nucleocapsid is orchestrated, and highlight an interaction site representing an important target for antivirals. DOI: http://dx.doi.org/10.7554/eLife.12627.001","publicationTypes":["JournalArticle"],"tldr":null},"tag":"EXP"},{"id":332,"details":{"paperId":"731dbc63f756b36bd8e9b1d09e521002426c05a7","externalIds":{"MAG":"1918318031","DOI":"10.1128/jvi.50.1.92-99.1984","CorpusId":"25235514","PubMed":"6699948"},"title":"Nucleotide sequence of the gene encoding respiratory syncytial virus matrix protein","abstract":"The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides upstream of the polyadenylate tail are conserved in all genes. There is no equivalent of the eucaryotic polyadenylation signal AAUAAA upstream of the polyadenylate tail. The matrix protein of 28,717 daltons has 256 amino acids. It is relatively basic and moderately hydrophobic. There are two clusters of hydrophobic amino acid residues in the C-terminal third of the protein that could potentially interact with the membrane components of the infected cell. The matrix protein has no homology with the matrix proteins of other negative-strand RNA viruses, implying that RS virus has undergone extensive evolutionary divergence. A second open reading frame potentially encoding a protein of 75 amino acids and partially overlapping the C terminus of the matrix protein was also identified.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The amino acid sequence of the matrix protein of the human respiratory syncytial virus was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene."}},"tag":"EXP"},{"id":47,"details":{"paperId":"0a78bc48a8d2d902eb58137da997524b66a4e715","externalIds":{"MAG":"2067296086","DOI":"10.1016/0042-6822(91)90532-G","CorpusId":"9229622","PubMed":"1840712"},"title":"Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt."}},"tag":"EXP"},{"id":336,"details":{"paperId":"3ce71537d58b6aae757353c9af6da39d8b11e0ab","externalIds":{"MAG":"1810552033","DOI":"10.1128/jvi.64.8.4007-4012.1990","CorpusId":"35263131","PubMed":"2164608"},"title":"O glycosylation of glycoprotein G of human respiratory syncytial virus is specified within the divergent ectodomain","abstract":"cDNAs encoding the G glycoprotein of respiratory syncytial virus and the hemagglutinin-neuraminidase (HN) glycoprotein of parainfluenza virus type 3 were modified by site-specific mutagenesis and restriction fragment replacement to encode chimeric proteins consisting of the cytoplasmic and transmembrane domains of one protein fused to the ectodomain of the other. In the case of the HN ectodomain attached to the G transmembrane and cytoplasmic domains, cell surface expression of the chimera was reduced. Otherwise, the presence of the heterologous transmembrane and cytoplasmic domains had little effect on the processing of the HN or G ectodomain, as assayed by the acquisition of N-linked and O-linked carbohydrates, transport to the cell surface and, in the case of HN, folding, oligomerization, and hemadsorption activity. These results showed that the synthesis and processing of each ectodomain did not require the homologous transmembrane and cytoplasmic domains. In particular, O glycosylation of the G protein was specified fully by its ectodomain, even though this domain is highly divergent among the respiratory syncytial virus antigenic subgroups. In addition, whereas the cytoplasmic and transmembrane domains of the G protein were relatively highly conserved, they were nonetheless fully replaceable without significantly affecting processing.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results showed that the synthesis and processing of each ectodomain did not require the homologous transmembrane and cytoplasmic domains, and O glycosylation of the G protein was specified fully by its ectodmain, even though this domain is highly divergent among the respiratory syncytial virus antigenic subgroups."}},"tag":"EXP"},{"id":218,"details":{"paperId":"53a06b0e0a827c7eec4402905a86a38b67a8e92d","externalIds":{"MAG":"2018546389","DOI":"10.1099/0022-1317-73-4-849","CorpusId":"11411093","PubMed":"1634876"},"title":"Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A.","abstract":"The post-translational maturation of the attachment G glycoprotein of human respiratory syncytial virus (RSV) was investigated. The G protein formed homo-oligomers which sedimented in sucrose gradients at the same rate as the fusion F protein tetramer. Oligomerization of the G protein was insensitive to carbonylcyanide m-chlorophenylhydrazine, showing that this step occurs in the endoplasmic reticulum prior to O-glycosylation which initiated in the trans-Golgi compartment. The sedimentation of the G protein oligomer was essentially unchanged by the subsequent addition of O-linked sugars. This indicated that their contribution to the M(r) of the G protein is less than that estimated by electrophoretic mobility. It also suggested that O-glycosylation is not an important determinant of G protein oligomerization and, by implication, of polypeptide folding. The G protein is palmitylated. In short labelling pulses, the G protein accumulated as two species of 48K and 50K which contained only N-linked sugars, whose difference in M(r) was due solely to an N-linked sugar, which both assembled into oligomers, but which differed in the rate of subsequent O-glycosylation. The G protein was not detectably O-glycosylated in the presence of monensin, confirming previous work. In the presence of brefeldin A (BFA), it accumulated as a partially O-glycosylated species (BFA-G) of 68K to 78K. But further analysis by chase incubations following BFA-washout, by lectin-binding, and by glycosidase treatment suggested that BFA-G was not a fully authentic processing intermediate. In particular, some of the O-linked side-chains of the BFA-G protein were found to be sialylated. Rather than being a normal step in processing, this sialylation probably was due to altered distribution or activity of sialyltransferases during BFA treatment and may have resulted in the premature termination of elongation of some of the O-linked side-chains. Thus, these studies (i) indicate that O-glycosylation of the G protein begins in the trans-Golgi compartment and (ii) suggest that O-glycosylation is completed in as a subsequent compartment, but this latter suggestion is complicated by the evidence that the BFA-G protein is not a fully authentic intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"O-glycosylation of the G protein begins in the trans-Golgi compartment and the suggestion that O-glyCosylation is completed in as a subsequent compartment is suggested, but this latter suggestion is complicated by the evidence that the BFA-G protein is not a fully authentic intermediate."}},"tag":"EXP"},{"id":392,"details":{"paperId":"0f984cef1db0705d603887ab4d131624af0adba1","externalIds":{"MAG":"1974037039","DOI":"10.1128/JVI.79.22.13943-13952.2005","CorpusId":"25265988","PubMed":"16254330"},"title":"Overexpression of the M2-2 Protein of Respiratory Syncytial Virus Inhibits Viral Replication","abstract":"ABSTRACT The M2-2 protein of respiratory syncytial virus (RSV) is involved in regulation of viral RNA transcription and replication. Encoded by the next-to-last gene of RSV, the M2-2 open reading frame (ORF) overlaps with the upstream M2-1 ORF, suggesting that the production of the M2-2 protein might be tightly regulated during virus replication. To evaluate the effect of M2-2 overexpression on RSV replication, the M2-2 gene was separated from M2-1 by leaving it at the position prior to the M2-1 or moving it to the promoter proximal position as an independent transcriptional unit in the RSV A2 genome. Although recombinant viruses bearing the shuffled M2-2 gene were recovered and expressed higher levels of M2-2, most of these viruses grew poorly in HEp-2 cells. Sequence analysis revealed that various mutations (substitution, insertion, and deletion) occurred in the M2-2 gene, resulting in reduced M2-2 activity as measured by the RSV minigenome system. Further examination of the M2-2 sequence and its function showed that either one of the first two AUG codons located at the 5′ end of M2-2 could be used to produce a functional M2-2 protein and that deletion of the first six amino acids from its N terminus or four amino acids from its C terminus greatly reduced its function. The effect of M2-2 protein on RSV replication was also studied by examining RSV replication in cells transiently expressing M2-2. The M2-2 protein expressed at a high level completely inhibited RSV replication. These results strongly suggested that the level of the M2-2 protein produced in the infected cells is critical to RSV replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results strongly suggested that the level of the M2-2 protein produced in the infected cells is critical to RSV replication, and that deletion of the first six amino acid from its N terminus or four amino acids from its C terminus greatly reduced its function."}},"tag":"EXP"},{"id":50,"details":{"paperId":"258779291ae0c2c68e5449dd506d6c33c0988905","externalIds":{"MAG":"2020517476","DOI":"10.1016/0168-1702(88)90090-1","CorpusId":"4223528","PubMed":"2837016"},"title":"Participation of cytoskeletal intermediate filaments in the infectious cycle of human respiratory syncytial virus (RSV).","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that RSV needs to interact with IF during its life cycle and that association of NP, and/or other viral components, with IF might then lead to cytoskeletal structures becoming unstable in RSV-infected cells."}},"tag":"EXP"},{"id":43,"details":{"paperId":"6e7638298567aeda74e61333904c6e0e06109a19","externalIds":{"PubMedCentral":"5840107","MAG":"2791852979","DOI":"10.1007/s40121-018-0188-z","CorpusId":"3719319","PubMed":"29470837"},"title":"Past, Present and Future Approaches to the Prevention and Treatment of Respiratory Syncytial Virus Infection in Children","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Management of RSV remains centered around prophylaxis with the monoclonal antibody palivizumab, which has proven effective in reducing RSV hospitalization in preterm infants, children with bronchopulmonary dysplasia, and infants with hemodynamically significant congenital heart disease."}},"tag":"OTHER"},{"id":351,"details":{"paperId":"a427f900829ee3ce3fb0e18f12ae18ca4eb5b228","externalIds":{"MAG":"2158608067","DOI":"10.1128/JVI.72.7.5610-5618.1998","CorpusId":"25031245","PubMed":"9621019"},"title":"Persistent Activation of RelA by Respiratory Syncytial Virus Involves Protein Kinase C, Underphosphorylated IκBβ, and Sequestration of Protein Phosphatase 2A by the Viral Phosphoprotein","abstract":"ABSTRACT Respiratory syncytial virus (RSV) activated the RelA (p65) subunit of nuclear factor kappa B (NF-κB) over many hours postinfection. The initial activation coincided with phosphorylation and degradation of IκBα, the cytoplasmic inhibitor of RelA. During persistent activation of NF-κB at later times in infection, syntheses of inhibitors IκBα as well as IκBβ were restored. However, the resynthesized IκBβ was in an underphosphorylated state, which apparently prevented inhibition of NF-κB. Use of specific inhibitors suggested that the pathway leading to the persistent—but not the initial—activation of NF-κB involved signaling through protein kinase C (PKC) and reactive oxygen intermediates of nonmitochondrial origin, whereas phospholipase C or D played little or no role. Thus, RSV infection led to the activation of NF-κB by a biphasic mechanism: a transient or early activation involving phosphorylation of the inhibitor IκB polypeptides, and a persistent or long-term activation requiring PKC and the generation of hypophosphorylated IκBβ. At least a part of the activation was through a novel mechanism in which the viral phosphoprotein P associated with but was not dephosphorylated by protein phosphatase 2A and thus sequestered and inhibited the latter. We postulate that this led to a net increase in the phosphorylation state of signaling proteins that are responsible for RelA activation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV infection led to the activation of NF-κB by a biphasic mechanism: a transient or early activation involving phosphorylation of the inhibitor IκB polypeptides, and a persistent or long-term activation requiring PKC and the generation of hypophosphorylated Iκbβ."}},"tag":"EXP"},{"id":11,"details":{"paperId":"74109675713eee0b7c34ca1ef7f1772a980321d9","externalIds":{"MAG":"1974838108","DOI":"10.1006/VIRO.1995.0013","CorpusId":"21440015","PubMed":"7491765"},"title":"Phosphorylation of Ser232 directly regulates the transcriptional activity of the P protein of human respiratory syncytial virus: phosphorylation of Ser237 may play an accessory role.","abstract":"The phosphoprotein P of human respiratory syncytial virus (RSV) was expressed in eukaryotic cells in phosphorylated form. Site-directed mutagenesis of the recombinant protein established Ser232 as the major site of phosphorylation in vivo. Phosphorylation of bacterially made P protein in vitro by purified casein kinase II (CKII) resulted in the phosphorylation of Ser237, whereas mainly Ser232 was phosphorylated by a crude cell extract. The P kinase activity in the cell extract exhibited properties characteristic of CKII. While the Ser232,237 to Ala double mutant was nearly completely defective for phosphorylation and transcription, phosphorylation at Ser232, through the use of appropriate P mutant or kinase, activated P protein. Phosphorylation of Ser237 restored activity only to the extent it facilitated phosphorylation of Ser232. Phosphate groups of P protein in RSV-infected cells were highly stable; inhibitors of protein serine phosphatases had no effect on the intracellular turnover of the phosphates. Highly purified viral polymerase L was transcriptionally active but devoid of P protein kinase activity. Thus, CKII-mediated phosphorylation of Ser232 appears to be the primary regulator of P protein activity while phosphorylation of Ser237 may be involved in a modulatory role under certain conditions.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"CKII-mediated phosphorylation of Ser232 appears to be the primary regulator of P protein activity while phosphorylated of Ser237 may be involved in a modulatory role under certain conditions."}},"tag":"EXP"},{"id":86,"details":{"paperId":"406403393d616e78841c94d9bee4be5f996128ef","externalIds":{"MAG":"2078592527","DOI":"10.1016/j.virol.2008.06.045","CorpusId":"40744938","PubMed":"18706669"},"title":"Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre, and Administration of LiCl to humans via aerosol should prevent H RSV infection, without secondary effects."}},"tag":"EXP"},{"id":249,"details":{"paperId":"4528b946530c345913ca0d96a267fbee64027bba","externalIds":{"MAG":"1986135139","DOI":"10.1099/VIR.0.82165-0","CorpusId":"24654655","PubMed":"17098979"},"title":"Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex.","abstract":"The human respiratory syncytial virus (HRSV) P protein is phosphorylated, with different turnover rates, at several serine (S) and threonine (T) residues. The role of phosphothreonines in viral RNA synthesis was studied by using P protein substitution variants and the HRSV-based minigenome pM/SH. By using liquid chromatography coupled to ion-trap mass spectrometry, it was found that P protein T108 was phosphorylated by addition of a high-turnover phosphate group. This phosphorylation occurs in P protein expressed transiently and during HRSV infection. The results suggest that phosphorylation at P protein T108 affects M2-1 transcriptional activities, because this modification prevents interaction between the P and M2-1 proteins. Therefore, P protein phosphorylation-dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"P protein phosphorylation-dephosphorylation at T108 could distinguish the role of the P protein in viral transcription and replication and affect M2-1 transcriptional activities."}},"tag":"EXP"},{"id":100,"details":{"paperId":"e63da11a438c572f570b9066959bbbc9473d8522","externalIds":{"MAG":"2065103350","DOI":"10.1016/j.virusres.2011.10.004","CorpusId":"28741683","PubMed":"22019509"},"title":"Phosphorylation of the human respiratory syncytial virus N protein provokes a decrease in viral RNA synthesis.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Y38 phosphorylation of the HRSV N protein modulates NC template activity probably by altering the interactions of the monomeric components of the NC."}},"tag":"EXP"},{"id":105,"details":{"paperId":"d595d20df6ae66132ca47c463ac06e7b695a3979","externalIds":{"MAG":"2195836881","DOI":"10.1016/j.virusres.2015.10.011","CorpusId":"33423950","PubMed":"26474524"},"title":"Phosphorylation of the human respiratory syncytial virus P protein mediates M2-2 regulation of viral RNA synthesis, a process that involves two P proteins.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that M2-2 mediated transcriptional inhibition is managed through P protein phosphorylation, and two types of P proteins participate in the process as components of RNA dependent RNA polymerase (RdRp)."}},"tag":"EXP"},{"id":49,"details":{"paperId":"8688ebe353405dcbabf8a892a6e70bb89bc5ba96","externalIds":{"MAG":"1983944049","DOI":"10.1016/0042-6822(92)90203-2","CorpusId":"21275328","PubMed":"1413513"},"title":"Polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (SH) protein: a conserved feature among human and bovine respiratory syncytial viruses.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A comparison of the deduced amino acid sequences of the human and bovine RS virus SH proteins indicated that a central hydrophobic region and the presence of potential N-linked glycosylation sites on either side of the central hydphobic region were conserved features that may be required for the polylactosaminoglycan modification of SH."}},"tag":"EXP"},{"id":459,"details":{"paperId":"2dc2488675594160336e67618feaeddeb346d24a","externalIds":{"PubMedCentral":"7575074","MAG":"3092038201","DOI":"10.1371/journal.ppat.1008987","CorpusId":"222236470","PubMed":"33031461"},"title":"Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection","abstract":"The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection details a critical understanding into the localization and activity of RSV L and the ribon DNAapsid complex during RSV infection."}},"tag":"EXP"},{"id":214,"details":{"paperId":"846659037a0178a2ea6bd30f00bf1cb789f56ddf","externalIds":{"MAG":"2149781357","DOI":"10.1099/0022-1317-72-12-3095","CorpusId":"20020551","PubMed":"1765771"},"title":"Post-translational processing and oligomerization of the fusion glycoprotein of human respiratory syncytial virus.","abstract":"The post-translational maturation of the fusion protein (F) of human respiratory syncytial virus was investigated. Chemical cross-linking experiments indicated that F forms homotetramers and provided evidence that the intermonomer contacts involve primarily the F1 subunit. Homooligomerization as measured by sedimentation in sucrose gradients was insensitive to carbonyl cyanide m-chlorophenylhydrazone, indicating that it occurs in the endoplasmic reticulum. Cleavage of the F0 precursor to yield the F1 and F2 subunits was blocked by monensin or brefeldin A, indicating that it takes place in distal cisternae of the trans Golgi compartment or in the more distal trans Golgi network. The F0 precursor was not detected at the cell surface in surface immunoprecipitation experiments, indicating that cleavage is intracellular. The appearance of the cleaved F1 protein at the cell surface was concurrent with that of the attachment glycoprotein (G); this and other information indicated that the type 2 membrane orientation of G is not obligatorily associated with a reduced transit rate. Examination of F maturation in the presence of tunicamycin provided evidence that its expression at the cell surface depends upon cleavage and not directly upon glycosylation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Examination of F maturation in the presence of tunicamycin provided evidence that its expression at the cell surface depends upon cleavage and not directly upon glycosylation, and indicated that the type 2 membrane orientation of G is not obligatorily associated with a reduced transit rate."}},"tag":"EXP"},{"id":166,"details":{"paperId":"9d8fcd9faf18d354d1210d0384caae90e701343c","externalIds":{"MAG":"2068160897","DOI":"10.1073/PNAS.92.25.11563","CorpusId":"40414559","PubMed":"8524804"},"title":"Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.","abstract":"Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It should be possible to introduce defined changes into infectious RSV using the M2(ORF1) protein, consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression."}},"tag":"EXP"},{"id":368,"details":{"paperId":"0223edd3c82767519007d8f6329441c081d2d041","externalIds":{"MAG":"2151818411","DOI":"10.1128/JVI.74.2.669-675.2000","CorpusId":"512225","PubMed":"10623728"},"title":"Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA","abstract":"ABSTRACT Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-l-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-l-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-l-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome."}},"tag":"EXP"},{"id":420,"details":{"paperId":"856ade56675365884c440b85de0e1a9c235a8ef2","externalIds":{"MAG":"1490759475","PubMedCentral":"156654","DOI":"10.1186/1471-2180-3-9","CorpusId":"1039785","PubMed":"12740026"},"title":"Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Profilin plays a cardinal role in RSV-mediated cell fusion and viral maturation, and interaction of profilin with the viral transcriptional proteins P and N may only nominally activate viral RNA-dependent RNA polymerase."}},"tag":"EXP"},{"id":99,"details":{"paperId":"ab316d1f88263ab160261be24b4e472714ee346d","externalIds":{"MAG":"1989959574","DOI":"10.1016/j.virusres.2011.09.020","CorpusId":"22720761","PubMed":"21963675"},"title":"Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV is notable for a historic, tragic pediatric vaccine failure involving a formalin-inactivated virus preparation that was evaluated in the 1960s and that was poorly protective and paradoxically primed for enhanced RSV disease."}},"tag":"OTHER"},{"id":509,"details":{"paperId":"7c8a2ef860c4cfb77bdeca914060df92e0a4ad82","externalIds":{"MAG":"2894248687","DOI":"10.4049/jimmunol.1800558","CorpusId":"52895272","PubMed":"30275049"},"title":"Proinflammatory Effects of Respiratory Syncytial Virus–Induced Epithelial HMGB1 on Human Innate Immune Cell Activation","abstract":"High mobility group box 1 (HMGB1) is a multifunctional nuclear protein that translocates to the cytoplasm and is subsequently released to the extracellular space during infection and injury. Once released, it acts as a damage-associated molecular pattern and regulates immune and inflammatory responses. Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in infants and elderly, for which no effective treatment or vaccine is currently available. This study investigated the effects of HMGB1 on cytokine secretion, as well as the involvement of NF-κB and TLR4 pathways in RSV-induced HMGB1 release in human airway epithelial cells (AECs) and its proinflammatory effects on several human primary immune cells. Purified HMGB1 was incubated with AECs (A549 and small alveolar epithelial cells) and various immune cells and measured the release of proinflammatory mediators and the activation of NF-κB and P38 MAPK. HMGB1 treatment significantly increased the phosphorylation of NF-κB and P38 MAPK but did not induce the release of cytokines/chemokines from AECs. However, addition of HMGB1 to immune cells did significantly induce the release of cytokines/chemokines and activated the NF-κB and P38 MAPK pathways. We found that activation of NF-κB accounted for RSV-induced HMGB1 secretion in AECs in a TLR4-dependent manner. These results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis. Therefore, blocking the proinflammatory function of HMGB1 may be an effective approach for developing novel therapeutics.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results indicated that HMGB1 secreted from AECs can facilitate the secretion of proinflammatory mediators from immune cells in a paracrine mechanism, thus promoting the inflammatory response that contributes to RSV pathogenesis."}},"tag":"EXP"},{"id":182,"details":{"paperId":"cfcf03fdeaaabc52e5017362b5d26e980457842a","externalIds":{"MAG":"2167787408","DOI":"10.1074/mcp.M110.001651","CorpusId":"25142489","PubMed":"20530633"},"title":"Protein Analysis of Purified Respiratory Syncytial Virus Particles Reveals an Important Role for Heat Shock Protein 90 in Virus Particle Assembly*","abstract":"In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of β-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The utility of proteomics is highlighted in facilitating both the authors' understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy."}},"tag":"EXP"},{"id":386,"details":{"paperId":"b7023b9a3709c64432ab9b64abd27be46f8aa539","externalIds":{"MAG":"2043022046","DOI":"10.1128/JVI.78.24.13717-13726.2004","CorpusId":"14674338"},"title":"Protein Kinase C-α Activity Is Required for Respiratory Syncytial Virus Fusion to Human Bronchial Epithelial Cells","abstract":"ABSTRACT Respiratory syncytial virus (RSV) infection activates protein kinase C (PKC), but the precise PKC isoform(s) involved and its role(s) remain to be elucidated. On the basis of the activation kinetics of different signaling pathways and the effect of various PKC inhibitors, it was reasoned that PKC activation is important in the early stages of RSV infection, especially RSV fusion and/or replication. Herein, the role of PKC-α during the early stages of RSV infection in normal human bronchial epithelial cells is determined. The results show that the blocking of PKC-α activation by classical inhibitors, pseudosubstrate peptides, or the overexpression of dominant-negative mutants of PKC-α in these cells leads to significantly decreased RSV infection. RSV induces phosphorylation, activation, and cytoplasm-to-membrane translocation of PKC-α. Also, PKC-α colocalizes with virus particles and is required for RSV fusion to the cell membrane. Thus, PKC-α could provide a new pharmacological target for controlling RSV infection.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The results show that the blocking of PKC-α activation by classical inhibitors, pseudosubstrate peptides, or the overexpression of dominant-negative mutants of PKP-α in normal human bronchial epithelial cells leads to significantly decreased RSV infection."}},"tag":"EXP"},{"id":472,"details":{"paperId":"0fda9c6dd680860a7a993f9a55d8b334e84f01b7","externalIds":{"MAG":"2327092997","DOI":"10.2174/187152612800100125","CorpusId":"34611074","PubMed":"22335497"},"title":"Protein-protein interactions in RSV assembly: potential targets for attenuating RSV strains.","abstract":"Respiratory syncytial virus (RSV) is the major respiratory pathogen of infants and children worldwide, with no effective treatment or vaccine available. Steady progress has been made in understanding the respiratory syncytial virus lifecycle and the consequences of infection, but some areas of RSV still remain poorly understood. Although many of the interactions between virus proteins that are required for efficient RSV assembly have been elucidated, many questions still remain regarding viral assembly, as well as the mechanisms of RSV budding. This review will summarise the current understanding of RSV assembly, including the various interactions between virus proteins and the involvement of cellular factors with a view to identifying possible attenuation and/or drug targets within the assembly pathway.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The current understanding of RSV assembly is summarized, including the various interactions between virus proteins and the involvement of cellular factors with a view to identifying possible attenuation and/or drug targets within the assembly pathway."}},"tag":"EXP"},{"id":171,"details":{"paperId":"feb687afbac2c9e0614788c135891571cbf3be40","externalIds":{"MAG":"2114630983","DOI":"10.1074/JBC.M102633200","CorpusId":"21029754","PubMed":"11418598"},"title":"Proteolytic Activation of Respiratory Syncytial Virus Fusion Protein","abstract":"The F (fusion) protein of the respiratory syncytial viruses is synthesized as an inactive precursor F0 that is proteolytically processed at the multibasic sequence KKRKRR136 into the subunits F1 and F2 by the cellular protease furin. This maturation process is essential for the F protein to gain fusion competence. We observed that proteolytic cleavage additionally occurs at another basic motif, RARR109 , that also meets the requirements for furin recognition. Cleavage at both sites leads to the removal from the polypeptide chain of a glycosylated peptide of 27 amino acids. When the sequence RARR109 was changed to NANR109 or to RANN109 by site-directed mutagenesis, cleavage by furin was completely prevented. Although the mutants were still processed at position Arg136, they did not show any syncytia formation. Proteolytic cleavage of the modified motifs was achieved by treatment of transfected cells with trypsin converting the F mutants into their fusogenic forms. Our findings indicate that both furin consensus sequences have to be cleaved in order to activate the fusion protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is observed that proteolytic cleavage additionally occurs at another basic motif, RARR109 , that also meets the requirements for furin recognition, and both furin consensus sequences have to be cleaved in order to activate the fusion protein."}},"tag":"EXP"},{"id":72,"details":{"paperId":"544e35512542f71c156a5e3913e8b44125ba95d8","externalIds":{"MAG":"2046041776","DOI":"10.1016/J.PEP.2007.09.017","CorpusId":"37112797","PubMed":"17997327"},"title":"Purification and characterization of recombinant human respiratory syncytial virus nonstructural protein NS1.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The availability of purified RSV NS1 will permit biochemical and structural investigations providing insight into the function of NS1 in viral replication and interferon antagonism."}},"tag":"EXP"},{"id":205,"details":{"paperId":"a207b00fe9962368558bb6dbf317b1186560001e","externalIds":{"MAG":"2123704395","DOI":"10.1099/0022-1317-66-3-409","CorpusId":"30684848","PubMed":"3838336"},"title":"Purification and characterization of the respiratory syncytial virus fusion protein.","abstract":"The fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody. Under various conditions the protein was recovered as a 145K dimer or a 70K monomer. The 70K monomer was composed of disulphide-linked fragments of 48K and 23K. Polyclonal rabbit serum produced to the dimerized fusion protein neutralized virus but did not inhibit fusion, while rabbit serum to the 2-mercaptoethanol-treated dimerized protein neutralized virus and inhibited fusion of infected cells. Only the latter serum strongly recognized the 23K fragment when studied by Western blot analysis.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody and recovered as a 145K dimer or a 70K monomer under various conditions."}},"tag":"EXP"},{"id":117,"details":{"paperId":"a5588266701dae8a87db46678659b5f2e163dbfe","externalIds":{"MAG":"2015298230","DOI":"10.1016/S0378-4347(99)00442-9","CorpusId":"31068458","PubMed":"10681046"},"title":"Purification of respiratory syncytial virus F and G proteins.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"No loss of antigenicity could be observed during this procedure as the highly purified viral proteins remain detectable by a set of monoclonal antibodies and specific antisera."}},"tag":"EXP"},{"id":426,"details":{"paperId":"bdce45fc16fbb70b05c054383aa9e1e6dd2e9bf5","externalIds":{"DOI":"10.1186/s12985-014-0191-2","CorpusId":"255955110"},"title":"Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid protein","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription."}},"tag":"EXP"},{"id":370,"details":{"paperId":"fc5bc20712c333ee19cb3b484099ce705524d5cd","externalIds":{"MAG":"2062889746","DOI":"10.1128/JVI.75.1.36-44.2001","CorpusId":"9264479","PubMed":"11119571"},"title":"RNA Sequences Involved in Transcriptional Termination of Respiratory Syncytial Virus","abstract":"ABSTRACT RNA signals at the ends of the genes of respiratory syncytial (RS) virus direct polyadenylation and termination of viral transcription. These gene ends contain two conserved regions, a pentanucleotide and a tract of uridylate (U) residues, separated by an A/U-rich central region that is less well conserved. The U tract is thought to be the template for polyadenylation of viral mRNAs by reiterative transcription. The cis-acting requirements for termination were investigated by mutagenesis of the matrix (M) gene end (3′-UCAAUUAUUUUUU-5′) in a dicistronic RNA replicon. Termination efficiencies were quantitated by intracellular metabolic labeling of monocistronic mRNAs and the dicistronic readthrough RNAs that result when termination fails to occur. All three regions of the gene end were necessary for termination. Mutation of each of the first 8 nucleotides of the M gene end to all other nucleotides showed that nucleotides 2 to 6 were important for termination and intolerant of change, whereas nucleotides 1 and 7 were tolerant of change. At position 8, A or U allowed termination, but G or C did not. Both the length and the position of the U tract were important for termination. U residues at positions 9 to 12 were necessary, while additional U residues at position 8, and especially position 13, enhanced termination efficiency. Altering the length of the central region abolished termination, suggesting that the position of the U tract with respect to the 3′-UCAAU-5′ sequence was critical. The termination efficiencies of each of the 10 genes of RS virus are different. Since transcription is obligatorily sequential and termination of each gene is required for transcription of the next gene downstream, these differences may contribute to gene regulation. In agreement with our data, the naturally occurring gene ends of RS virus that terminate inefficiently have short U tracts or other sequence features that correlated with decreased termination when similar mutations were analyzed in RNA replicons.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The naturally occurring gene ends of RS virus that terminate inefficiently have short U tracts or other sequence features that correlated with decreased termination when similar mutations were analyzed in RNA replicons."}},"tag":"EXP"},{"id":451,"details":{"paperId":"e6650167b0764499b35c7e1b1eedb900beb69102","externalIds":{"PubMedCentral":"5760109","MAG":"2778218941","DOI":"10.1371/journal.ppat.1006803","CorpusId":"6717662","PubMed":"29281742"},"title":"RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V","abstract":"The large polymerase subunit (L) of non-segmented negative strand RNA viruses transcribes viral mRNAs and replicates the viral genome. Studies with VSV have shown that conserved region V (CRV) of the L protein is part of the capping domain. However, CRV folds over and protrudes into the polymerization domain, suggesting that it might also have a role in RNA synthesis. In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D. Effects were analyzed using cell-based minigenome and in vitro biochemical assays. Several amino acid substitutions inhibited production of capped, full-length mRNA and instead resulted in accumulation of short transcripts of approximately 40 nucleotides in length, confirming that RSV CRV has a role in capping. In addition, all six variants tested were either partially or completely defective in RNA replication. This was due to an inability of the polymerase to efficiently elongate the RNA within the promoter region. BI-D also inhibited transcription and replication. In this case, polymerase elongation activity within the promoter region was enhanced, such that the small RNA transcribed from the promoter was not released and instead was elongated past the first gene start signal. This was accompanied by a decrease in mRNA initiation at the first gene start signal and accumulation of aberrant RNAs of varying length. Thus, in addition to its function in mRNA capping, conserved region V modulates the elongation properties of the polymerase to enable productive transcription and replication to occur.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In this study, the role of respiratory syncytial virus (RSV) CRV was evaluated using single amino acid substitutions and a small molecule inhibitor called BI-D to enable productive transcription and replication to occur."}},"tag":"EXP"},{"id":345,"details":{"paperId":"fd79be8daee770f69f2cf321ebdf0ca7e9e45849","externalIds":{"MAG":"2137902026","DOI":"10.1128/jvi.70.8.5075-5082.1996","CorpusId":"20589076","PubMed":"8764015"},"title":"RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end","abstract":"Genome analogs (\"minigenomes\") of Sendai and measles viruses replicate efficiently only if their nucleotide length is an even multiple of six, a requirement called the rule of six (P. Calain and L. Roux, J. Virol. 67:4822-4830, 1993; M. S. Sidhu, J. Chan, K. Kaelin, P. Spielhofer, F. Radecke, H. Schneider, M. Masurekar, P. C. Dowling, M. A. Billeter, and S. A. Udem, Virology 208:800-807, 1995). The existence of a comparable requirement was tested for respiratory syncytial virus (RSV), which also is a member of family Paramyxoviridae and whose natural genome length also is a multiple of six. An internally truncated analog of RSV positive-sense replicative intermediate RNA (antigenome) bearing the chloramphenicol acetyltransferase gene as a reporter was synthesized from cDNA in vitro. This RNA was transfected into cells which were infected with RSV as a helper. Miniantigenomic RNA was indistinguishable from previously studied negative-sense minigenome RNA in its ability to participate in transcription, RNA replication, and incorporation into transmissible particles. Sixteen miniantigenomes which were of slightly different lengths and which in aggregate represented multiples of a wide range of integers including 1 to 15 were constructed. During transfection and two serial passages, the various miniantigenomes were essentially indistinguishable with regard to the efficiency of transcription, RNA replication, and packaging into transmissible particles. Progeny minigenomes of six different mutants were recovered postpassage, copied into cDNA, cloned, and sequenced completely. The length of each of these RNAs was found to have remained unchanged during replication and passage. Thus, RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function. Each of the in vitro-synthesized miniantigenomes used in transfection contained a nonviral extension of three nucleotides, GGG, on the 5' (leader) end contributed by the T7 promoter. The termini of the recovered minigenomes were examined for five mutants by RNA circularization followed by cDNA synthesis, amplification, cloning, and sequencing. Unexpectedly, each recovered minigenome contained the complement of this nonviral extension on the 3' (leader) end, showing that it had been faithfully copied and maintained during RNA replication and passage. The nonviral trinucleotide did not appear to affect the activity of the template.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function."}},"tag":"EXP"},{"id":342,"details":{"paperId":"4f16a608c080ff85af9db2c6e75805747f05e0fb","externalIds":{"MAG":"1553990265","DOI":"10.1128/jvi.69.9.5677-5686.1995","CorpusId":"11967198","PubMed":"7637014"},"title":"RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA","abstract":"Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was \"rescued,\" as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini."}},"tag":"EXP"},{"id":74,"details":{"paperId":"2e20229dc19dc9d7e75916c304681e119e327c43","externalIds":{"MAG":"3082066283","DOI":"10.1016/j.str.2020.08.007","CorpusId":"221478465","PubMed":"32877647"},"title":"RSV M2-1 Protein in Complex with RNA: Old Questions Are Answered and a New One Emerges.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The crystal structure of M2-1 in complex with RNA is described, which provides new insight into the RSV transcription mechanism but also raises an intriguing question regarding M1-1's function."}},"tag":"EXP"},{"id":104,"details":{"paperId":"d6649db26d59c61cdae7b0bb7a3eb8c56b775d0d","externalIds":{"MAG":"1994701842","DOI":"10.1016/j.virusres.2015.04.018","CorpusId":"23266451","PubMed":"25937519"},"title":"RSV P-protein impairs extrinsic apoptosis pathway in a macrophage-like cell line persistently infected with respiratory syncytial virus.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that P-protein impaired the extrinsic pathway of apoptosis in cells persistently infected with RSV, contributing to the understanding of the mechanism by which viral proteins subvert the extrINSic apoptosis process."}},"tag":"EXP"},{"id":499,"details":{"paperId":"72609538f55fa97255eec0161234d070567f26f4","externalIds":{"PubMedCentral":"7232410","MAG":"3020686985","DOI":"10.3390/v12040472","CorpusId":"216130663","PubMed":"32331282"},"title":"RSV Reprograms the CDK9•BRD4 Chromatin Remodeling Complex to Couple Innate Inflammation to Airway Remodeling","abstract":"Respiratory syncytial virus infection is responsible for seasonal upper and lower respiratory tract infections worldwide, causing substantial morbidity. Self-inoculation of the virus into the nasopharynx results in epithelial replication and distal spread into the lower respiratory tract. Here, respiratory syncytial virus (RSV) activates sentinel cells important in the host inflammatory response, resulting in epithelial-derived cytokine and interferon (IFN) expression resulting in neutrophilia, whose intensity is associated with disease severity. I will synthesize key findings describing how RSV replication activates intracellular NFκB and IRF signaling cascades controlling the innate immune response (IIR). Recent studies have implicated a central role for Scg1a1+ expressing progenitor cells in IIR, a cell type uniquely primed to induce neutrophilic-, T helper 2 (Th2)-polarizing-, and fibrogenic cytokines that play distinct roles in disease pathogenesis. Molecular studies have linked the positive transcriptional elongation factor-b (P-TEFb), a pleiotrophic chromatin remodeling complex in immediate-early IIR gene expression. Through intrinsic kinase activity of cyclin dependent kinase (CDK) 9 and atypical histone acetyl transferase activity of bromodomain containing protein 4 (BRD4), P-TEFb mediates transcriptional elongation of IIR genes. Unbiased proteomic studies show that the CDK9•BRD4 complex is dynamically reconfigured by the innate response and targets TGFβ-dependent fibrogenic gene networks. Chronic activation of CDK9•BRD4 mediates chromatin remodeling fibrogenic gene networks that cause epithelial mesenchymal transition (EMT). Mesenchymal transitioned epithelial cells elaborate TGFβ and IL6 that function in a paracrine manner to expand the population of subepithelial myofibroblasts. These findings may account for the long-term reduction in pulmonary function in children with severe lower respiratory tract infection (LRTI). Modifying chromatin remodeling properties of the CDK9•BRD4 coactivators may provide a mechanism for reducing post-infectious airway remodeling that are a consequence of severe RSV LRTIs.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Modifying chromatin remodeling properties of the CDK9•BRD4 coactivators may provide a mechanism for reducing post-infectious airway remodeling that are a consequence of severe RSV LRTIs, and may account for the long-term reduction in pulmonary function in children with severe lower respiratory tract infection (LRTI)."}},"tag":"EXP"},{"id":406,"details":{"paperId":"7832a97a866dd107d9dac2af20981a93c8ec06c5","externalIds":{"PubMedCentral":"8321853","DOI":"10.1152/ajplung.00118.2021","CorpusId":"234793822","PubMed":"34010080"},"title":"RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration","abstract":"The airway epithelium’s ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract’s normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in day 7 naphthalene + RSV animals, which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A key mechanism by which RSV infection delays wound healing is suggested, which involves attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions."}},"tag":"EXP"},{"id":139,"details":{"paperId":"46d5a5b3b8968f9a0bf4811aabeb91f2eb91e88d","externalIds":{"DOI":"10.1038/s41467-017-00732-z","CorpusId":"256636726"},"title":"RSV glycoprotein and genomic RNA dynamics reveal filament assembly prior to the plasma membrane","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is discovered that plasma membrane-bound respiratory syncytial virus G rapidly recycles from the membrane via clathrin-mediated endocytosis and assembly with genomic ribonucleoproteins and caveolae-associated vesicles prior to re-insertion into the plasma membrane."}},"tag":"EXP"},{"id":453,"details":{"paperId":"6f08722392b29f032f73557746033c2061cb480a","externalIds":{"PubMedCentral":"5847313","MAG":"2788158824","DOI":"10.1371/journal.ppat.1006920","CorpusId":"4305020","PubMed":"29489893"},"title":"RSV hijacks cellular protein phosphatase 1 to regulate M2-1 phosphorylation and viral transcription","abstract":"Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P–PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P–PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M1-1, and that only dephosphorylated M 2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function."}},"tag":"EXP"},{"id":447,"details":{"paperId":"1ee85221e19d7bece6a97c4f58e5b9bd9ad5238d","externalIds":{"PubMedCentral":"4470918","MAG":"647519093","DOI":"10.1371/journal.ppat.1004978","CorpusId":"27372773","PubMed":"26083387"},"title":"RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo","abstract":"Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5b fl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5b fl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5b fl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In vivo and in vitro results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease."}},"tag":"EXP"},{"id":419,"details":{"paperId":"52cdc185861eac01e974e2a6cbd62fba2a109a6e","externalIds":{"MAG":"2010411959","DOI":"10.1172/JCI72948","CorpusId":"3370388","PubMed":"24713657"},"title":"RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction.","abstract":"Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease.","publicationTypes":["JournalArticle","ClinicalTrial"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that RSV NS2 is a potential therapeutic target for reducing the severity of distal airway disease and promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of thedistal airways."}},"tag":"EXP"},{"id":82,"details":{"paperId":"17aae21f17f08256c81a369b4a06b8d498f54806","externalIds":{"MAG":"2022394822","DOI":"10.1016/J.VIROL.2005.08.012","CorpusId":"27754451","PubMed":"16153673"},"title":"RSV-induced prostaglandin E2 production occurs via cPLA2 activation: role in viral replication.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV infection strongly induces PGE2 secretion, in a time- and replication-dependent manner, through increased cyclooxygenase-2 (COX-2) expression, which occurs independently from viral or cellular protein synthesis."}},"tag":"EXP"},{"id":478,"details":{"paperId":"290170bf365e7e74e828930f298be2917a29d18b","externalIds":{"PubMedCentral":"8913501","DOI":"10.3389/fcimb.2022.858629","CorpusId":"247085590","PubMed":"35281439"},"title":"Receptors for Respiratory Syncytial Virus Infection and Host Factors Regulating the Life Cycle of Respiratory Syncytial Virus","abstract":"Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections and responsible for a large proportion of mortality in children and the elderly. There are no licensed vaccines available to date. Prophylaxis and therapeutic RSV-specific antibodies are limited to populations at high risk owing to high cost and uncertain clinical value. Receptors and host factors are two determinants important for virus entry and establishment of infection in vivo. The identification and understanding of viral receptors and host factors can help us to gain insight into the pathogenesis of RSV infection. Herein, we reviewed receptors and host factors that have been reported thus far. RSV could bind to CX3C chemokine receptor 1 and heparan sulfate proteoglycans via the G protein, and to nucleolin, insulin-like growth factor-1 receptor, epidermal growth factor, and intercellular adhesion molecule-1 via the F protein. Seven host restriction factors and 13 host factors essential for RSV infection were reviewed. We characterized the functions and their roles in the life cycle of RSV, trying to provide an update on the information of RSV-related receptors and host factors.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Seven host restriction factors and 13 host factors essential for RSV infection were reviewed and characterized the functions and their roles in the life cycle of RSV, trying to provide an update on the information ofRSV-related receptors and host factors."}},"tag":"EXP"},{"id":442,"details":{"paperId":"e407605948be54c71e7725d9eba5e753c1f70560","externalIds":{"MAG":"2339488556","PubMedCentral":"4481108","DOI":"10.1371/journal.pone.0130829","CorpusId":"14498915","PubMed":"26107504"},"title":"Recombinant Soluble Respiratory Syncytial Virus F Protein That Lacks Heptad Repeat B, Contains a GCN4 Trimerization Motif and Is Not Cleaved Displays Prefusion-Like Characteristics","abstract":"The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble RSV F protein resulted in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate."}},"tag":"EXP"},{"id":347,"details":{"paperId":"b0ccb31531ecf42ee7b2e280366b7f1479438765","externalIds":{"MAG":"1919761071","DOI":"10.1128/jvi.71.12.8973-8982.1997","CorpusId":"20247433","PubMed":"9371553"},"title":"Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse","abstract":"The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that the small hydrophobic protein SH of human respiratory syncytial virus seems to be completely dispensable for growth in tissue culture and fusion function, and when inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract."}},"tag":"EXP"},{"id":109,"details":{"paperId":"9a8e12fb98910f561344cfead396570060a37802","externalIds":{"MAG":"2056520017","DOI":"10.1016/S0014-5793(00)01171-6","CorpusId":"45011705","PubMed":"10675554"},"title":"Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Purified human respiratory syncytial virus (HRSV) P phosphoprotein from transfected HEp‐2 cells is able to oligomerize forming tetramers, but dephosphorylated P protein, produced in bacteria, is unable to oligomersize."}},"tag":"EXP"},{"id":511,"details":{"paperId":"8778dac1ce0c4eb71c1320913ecdd5365e6dfe9f","externalIds":{"MAG":"2104619464","DOI":"10.4167/JBV.2014.44.3.283","CorpusId":"86669326"},"title":"Regulation of Host Cell Signaling Pathways by Respiratory Syncytial Virus Nonstructural Protein NS1 and NS2","abstract":"Human Respiratory Syncytial virus (hRSV) is a leading cause of severe lower respiratory tract diseases in the pediatric population.hRSV frequently causes severe morbidity and mortality in high risk groups including infants with congenital heart disease and the immunosuppressed patients. Although hRSV is recognized as a major public health threat and economic burden worldwide, there is no licensed vaccine and effective therapeutic agent. Viral nonstructural (NS) proteins have been known to play multiple functions for efficient viral replication and pathogenesis. Especially, diverse functions of influenza A virus NS1 have been extensively studies. Recent studies demonstrated that NS1 and NS2 of RSV also exert diverse functions to modulate cellular environment and antiviral immune responses. Since NS proteins of RSV are required for efficient replication and pathogenesis, NS mutant viruses have been tested as live-attenuated vaccines. This review will outline the recent progress in understanding the various functions of RSV NS1 and NS2.","publicationTypes":["Review"],"tldr":{"model":"tldr@v2.0.0","text":"This review will outline the recent progress in understanding the various functions of RSV NS1 and NS2."}},"tag":"EXP"},{"id":371,"details":{"paperId":"035f5bdf95b9e274d8cd2d08fd1a2c7495e61c87","externalIds":{"MAG":"2066283741","DOI":"10.1128/JVI.75.23.11328-11335.2001","CorpusId":"13057584","PubMed":"11689613"},"title":"Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability","abstract":"ABSTRACT The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys3-His1motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using β-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys3-His1motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys3-His1 motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys3-His1 motif. This indicates that the Cys3-His1 motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length β-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the Cterminus, which indicates that the Cys3-His1 motif is critical for M1-1 function and for RSV replication."}},"tag":"EXP"},{"id":10,"details":{"paperId":"7a1889f9bf97c8da572bb80122905783a549fdbb","externalIds":{"MAG":"2044724234","DOI":"10.1006/VIRO.1994.1624","CorpusId":"46345702","PubMed":"7975205"},"title":"Requirement of casein kinase II-mediated phosphorylation for the transcriptional activity of human respiratory syncytial viral phosphoprotein P: transdominant negative phenotype of phosphorylation-defective P mutants.","abstract":"The transcription complex of the human respiratory syncytial virus was biochemically dissected and reconstituted in vitro with purified viral macromolecules. The minimal complex consisted of the viral N-RNA template, viral phosphoprotein (P), and the large protein (L) along with host cellular factor(s), possibly actin. Active transcription could also be reconstituted using bacterially synthesized recombinant P protein provided the P protein was phosphorylated by cellular casein kinase II. Elimination of phosphorylation by inhibition of CKII or by mutation of the Ser residue at position 237 of the P protein also abrogated RSV transcription. In addition, the phosphorylation-defective P mutants exhibited a trans-dominant negative phenotype, consistent with the finding that the mutant proteins bound to the N-RNA template as efficiently as the wild type. Once engaged in transcription, however, the wild-type P protein became refractory to trans-inhibition by the mutant.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The phosphorylation-defective P mutants exhibited a trans-dominant negative phenotype, consistent with the finding that the mutant proteins bound to the N-RNA template as efficiently as the wild type, however, the wild-type P protein became refractory to trans-inhibition by the mutant."}},"tag":"EXP"},{"id":409,"details":{"paperId":"0fa4728e5e8a22c5a6f896ea9892844ac39b0220","externalIds":{"PubMedCentral":"3175114","MAG":"1999214411","DOI":"10.1155/2011/343408","CorpusId":"4025298","PubMed":"21931576"},"title":"Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells","abstract":"Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A novel role to the F and G proteins in viral protein localization and assembly is attributed to the glycoproteins of HRSV."}},"tag":"EXP"},{"id":98,"details":{"paperId":"134fd02e07586a1030ad34efdbbbd89cffc0a890","externalIds":{"MAG":"2041508323","DOI":"10.1016/j.virusres.2007.11.013","CorpusId":"46699034","PubMed":"18179840"},"title":"Residues in human respiratory syncytial virus P protein that are essential for its activity on RNA viral synthesis.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization, and compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect."}},"tag":"EXP"},{"id":383,"details":{"paperId":"02798fd9ae657828df8cb4fe79820c9e82cf47a7","externalIds":{"MAG":"2128293348","DOI":"10.1128/JVI.77.8.4609-4616.2003","CorpusId":"28321378","PubMed":"12663767"},"title":"Respiratory Syncytial Virus (RSV) Fusion Protein Subunit F2, Not Attachment Protein G, Determines the Specificity of RSV Infection","abstract":"ABSTRACT Human respiratory syncytial virus (HRSV) and bovine RSV (BRSV) infect human beings and cattle in a species-specific manner. We have here analyzed the contribution of RSV envelope proteins to species-specific entry into cells. In contrast to permanent cell lines, primary cells of human or bovine origin, including differentiated respiratory epithelia, peripheral blood lymphocytes, and macrophages, showed a pronounced species-specific permissivity for HRSV and BRSV infection, respectively. Recombinant BRSV deletion mutants lacking either the small hydrophobic (SH) protein gene or both SH and the attachment glycoprotein (G) gene retained their specificity for bovine cells, whereas corresponding mutants carrying the HRSV F gene specifically infected human cells. To further narrow the responsible region of F, two reciprocal chimeric F constructs were assembled from BRSV and HRSV F1 and F2 subunits. The specificity of recombinant RSV carrying only the chimeric F proteins strictly correlated with the origin of the membrane-distal F2 domain. A contribution of G to the specificity of entry could be excluded after reintroduction of BRSV or HRSV G. Virus with F1 and G from BRSV and with only F2 from HRSV specifically infected human cells, whereas virus expressing F1 and G from HRSV and F2 from BRSV specifically infected bovine cells. The introduction of G enhanced the infectivities of both chimeric viruses to equal degrees. Thus, the role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans. The identification of the F2 subunit as the determinant of RSV host cell specificity facilitates identification of virus receptors and should allow for development of reagents specifically interfering with RSV entry.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans."}},"tag":"EXP"},{"id":197,"details":{"paperId":"447a3da06a7c6b20f6cbe3e75adfe5242df5ba8d","externalIds":{"MAG":"2588070134","DOI":"10.1093/infdis/jix070","CorpusId":"205138664","PubMed":"28199704"},"title":"Respiratory Syncytial Virus (RSV) Infects CD4+ T Cells: Frequency of Circulating CD4+ RSV+ T Cells as a Marker of Disease Severity in Young Children","abstract":"Background\nAlthough human airway epithelial cells are the main target of respiratory syncytial virus (RSV), it also infects immune cells, such as macrophages and B cells. Whether T cells are permissive to RSV infection is unknown. We sought to analyze the permissiveness of CD4+ T cells to RSV infection.\n\n\nMethods\nCD4+ and CD8+ T cells from cord blood, healthy young children, and adults were challenged by RSV or cocultured with infected HEp-2 cells. Infection, phenotype, and cytokine production by T cells were analyzed by flow cytometry or enzyme-linked immunosorbent assay. Expression of RSV antigens by circulating CD4+ T cells from infected children was analyzed by flow cytometry, and disease severity was defined by standard criteria.\n\n\nResults\nCD4+ and CD8+ T cells were productively infected by RSV. Infection decreased interleukin 2 and interferon γ production as well as the expression of CD25 and Ki-67 by activated CD4+ T cells. Respiratory syncytial virus antigens were detected in circulating CD4+ and CD8+ T cells during severe RSV infection of young children. Interestingly, the frequency of CD4+ RSV+ T cells positively correlated with disease severity.\n\n\nConclusions\nRespiratory syncytial virus infects CD4+ and CD8+ T cells and compromises T-cell function. The frequency of circulating CD4+ RSV+ T cells might represent a novel marker of severe infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Interestingly, the frequency of CD4+ RSV+ T cells positively correlated with disease severity, and might represent a novel marker of severe infection."}},"tag":"EXP"},{"id":289,"details":{"paperId":"5d65b46fc58f93727df5dd4b59ad6664f78cd17e","externalIds":{"MAG":"2136490336","DOI":"10.1128/JVI.00671-08","CorpusId":"5879481","PubMed":"19019963"},"title":"Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Using knockout mice, it is demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES)."}},"tag":"EXP"},{"id":314,"details":{"paperId":"e63217a541e3d2c5e47f65a6085b1fe1e6e2891e","externalIds":{"PubMedCentral":"8103688","DOI":"10.1128/JVI.02333-20","CorpusId":"231753049","PubMed":"33504607"},"title":"Respiratory Syncytial Virus Activates Rab5a To Suppress IRF1-Dependent Lambda Interferon Production, Subverting the Antiviral Defense of Airway Epithelial Cells","abstract":"This study highlights the important role of Rab5a in respiratory syncytial virus (RSV) infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity and attenuates inflammation of the airway, which suggests that Rab5a is a powerful potential target for novel therapeutics against RSV infection. ABSTRACT The limited antiviral options and lack of an effective vaccine against human respiratory syncytial virus (RSV) highlight the need for a novel antiviral therapy. One alternative is to identify and target the host factors required for viral infection. Here, using RNA interference to knock down Rab proteins, we provide multiple lines of evidence that Rab5a is required for RSV infection: (i) Rab5a is upregulated both in RSV A2-infected A549 cells and RSV A2-challenged BALB/c mouse airway epithelial cells at early infection phase; (ii) short hairpin RNA (shRNA)-mediated knockdown of Rab5a is associated with reduced lung pathology in RSV A2-challenged mice; (iii) Rab5a expression is correlated with disease severity of RSV infection of infants. Knockdown of Rab5a increases lambda interferon (IFN-λ) production by mediating interferon regulatory factor 1 (IRF1) nuclear translocation. Our results highlight a new role for Rab5a in RSV infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity, which suggests that Rab5a is a potential target for novel therapeutics against RSV infection. IMPORTANCE This study highlights the important role of Rab5a in respiratory syncytial virus (RSV) infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity and attenuates inflammation of the airway, which suggests that Rab5a is a powerful potential target for novel therapeutics against RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This study highlights the important role of Rab5a in respiratory syncytial virus (RSV) infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity and attenuates inflammation of the airway, which suggests that Rab5A is a powerful potential target for novel therapeutics against RSV infections."}},"tag":"EXP"},{"id":436,"details":{"paperId":"8d063653ec169b742b76ffe8b3a6f6574921ccbc","externalIds":{"PubMedCentral":"3396619","MAG":"2052492915","DOI":"10.1371/journal.pone.0040826","CorpusId":"9832816","PubMed":"22808269"},"title":"Respiratory Syncytial Virus Assembles into Structured Filamentous Virion Particles Independently of Host Cytoskeleton and Related Proteins","abstract":"Respiratory syncytial virus (RSV) is a single-stranded RNA virus that assembles into viral filaments at the cell surface. Virus assembly often depends on the ability of a virus to use host proteins to accomplish viral tasks. Since the fusion protein cytoplasmic tail (FCT) is critical for viral filamentous assembly, we hypothesized that host proteins important for viral assembly may be recruited by the FCT. Using a yeast two-hybrid screen, we found that filamin A interacted with FCT, and mammalian cell experiments showed it localized to viral filaments but did not affect viral replication. Furthermore, we found that a number of actin-associated proteins also were excluded from viral filaments. Actin or tubulin cytoskeletal rearrangement was not necessary for F trafficking to the cell surface or for viral assembly into filaments, but was necessary for optimal viral replication and may be important for anchoring viral filaments. These findings suggest that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that RSV assembly into filaments occurs independently of actin polymerization and that viral proteins are the principal drivers for the mechanical tasks involved with formation of complex, structured RSV filaments at the host cell plasma membrane."}},"tag":"EXP"},{"id":311,"details":{"paperId":"0e38284c7c1ec0afc0d559e50eda22ea82a92f8d","externalIds":{"MAG":"2180865451","DOI":"10.1128/JVI.02140-15","CorpusId":"1415471","PubMed":"26468535"},"title":"Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein","abstract":"ABSTRACT Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. IMPORTANCE RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought."}},"tag":"EXP"},{"id":438,"details":{"paperId":"6a968a1367365addddde11f8a42f8cb873278ba7","externalIds":{"PubMedCentral":"4102526","MAG":"2037036613","DOI":"10.1371/journal.pone.0102368","CorpusId":"18559313","PubMed":"25033192"},"title":"Respiratory Syncytial Virus Can Infect Basal Cells and Alter Human Airway Epithelial Differentiation","abstract":"Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63+ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium and might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases."}},"tag":"EXP"},{"id":288,"details":{"paperId":"6256d69a3819421a10879bcaad0bb4f85ba56af4","externalIds":{"MAG":"2151131150","DOI":"10.1128/JVI.00643-06","CorpusId":"35266656","PubMed":"17005642"},"title":"Respiratory Syncytial Virus F Envelope Protein Associates with Lipid Rafts without a Requirement for Other Virus Proteins","abstract":"ABSTRACT Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.","publicationTypes":["JournalArticle","Study"],"tldr":{"model":"tldr@v2.0.0","text":"Data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein, which intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins."}},"tag":"EXP"},{"id":376,"details":{"paperId":"6fc8bebec9088a2713c4d6762ea8b914171f7622","externalIds":{"MAG":"2168766994","DOI":"10.1128/JVI.76.3.1163-1170.2002","CorpusId":"31204605","PubMed":"11773392"},"title":"Respiratory Syncytial Virus Fusion Protein Mediates Inhibition of Mitogen-Induced T-Cell Proliferation by Contact","abstract":"ABSTRACT Human respiratory syncytial virus (HRSV) and bovine respiratory syncytial virus (BRSV) are major pathogens in infants and calves, respectively. Experimental BRSV infection of calves and lambs is associated with lymphopenia and a reduction in responsiveness of peripheral blood lymphocytes (PBLs) to mitogens ex vivo. In this report, we show that in vitro mitogen-induced proliferation of PBLs is inhibited after contact with RSV-infected and UV-inactivated cells or with cells expressing RSV envelope proteins on the cell surface. The protein responsible was identified as the RSV fusion protein (F), as cells infected with a recombinant RSV expressing F as the single envelope protein or cells transfected with a plasmid encoding F were able to induce this effect. Thus, direct contact with RSV F is necessary and sufficient to inhibit proliferation of PBLs. Interestingly, F derived from HRSV was more efficient in inhibiting human PBL proliferation, while F from BRSV was more efficient in inhibiting bovine PBLs. Since various T-cell activation markers were upregulated after presenter cell contact, T lymphocytes are viable and may still be activated by mitogen. However, a significant fraction of PBLs were delayed or defective in G0/G1 to S-phase transit.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that in vitro mitogen-induced proliferation of PBLs is inhibited after contact with RSV-infected and UV-inactivated cells or with cells expressing RSV envelope proteins on the cell surface."}},"tag":"EXP"},{"id":440,"details":{"paperId":"c1af12682a525fbb372637cb734258dabe342e92","externalIds":{"MAG":"1971749790","PubMedCentral":"4391750","DOI":"10.1371/journal.pone.0124082","CorpusId":"2812475","PubMed":"25856628"},"title":"Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils","abstract":"Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is proposed that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis."}},"tag":"EXP"},{"id":306,"details":{"paperId":"80bdabc2441031ba947037a135746893fab8cb3d","externalIds":{"MAG":"2128972608","DOI":"10.1128/JVI.01741-13","CorpusId":"1202939","PubMed":"24089561"},"title":"Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)2 form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development."}},"tag":"EXP"},{"id":506,"details":{"paperId":"62275f4dec007ef67caa578cbf06c545d842a60f","externalIds":{"MAG":"1660139323","DOI":"10.4049/jimmunol.176.3.1600","CorpusId":"20139668","PubMed":"16424189"},"title":"Respiratory Syncytial Virus G Protein and G Protein CX3C Motif Adversely Affect CX3CR1+ T Cell Responses","abstract":"Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking. The respiratory syncytial virus (RSV) G protein has a CX3C chemokine motif that can bind CX3CR1 and modify CXCL1-mediated responses. In this study, we show that expression of the RSV G protein or the G protein CX3C motif during infection is associated with reduced CX3CR1+ T cell trafficking to the lung, reduced frequencies of RSV-specific, MHC class I-restricted IFN-γ-expressing cells, and lower numbers of IL-4- and CX3CL1-expressing cells. In addition, we show that CX3CR1+ cells constitute a major component of the cytotoxic response to RSV infection. These results suggest that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection."}},"tag":"EXP"},{"id":360,"details":{"paperId":"a758245b421b7d01073d67a44272a59e6f81c5b9","externalIds":{"MAG":"2102809170","DOI":"10.1128/JVI.73.9.7099-7107.1999","CorpusId":"26066840","PubMed":"10438795"},"title":"Respiratory Syncytial Virus G and/or SH Protein Alters Th1 Cytokines, Natural Killer Cells, and Neutrophils Responding to Pulmonary Infection in BALB/c Mice","abstract":"ABSTRACT BALB/c mice sensitized to vaccinia virus expressed G protein of respiratory syncytial virus (RSV) develop a Th2-type cytokine response and pulmonary eosinophilia when challenged with live RSV. In this study, BALB/c mice were immunized or challenged with an RSV mutant lacking the G and SH proteins or with DNA vaccines coding for RSV G or F protein. F or G protein DNA vaccines were capable of sensitizing for pulmonary eosinophilia. The absence of the G and/or SH protein in the infecting virus resulted in a consistent increase both in pulmonary natural killer cells and in gamma interferon and tumor necrosis factor expression, as well as, with primary infection, a variable increase in neutrophils and CD11b+ cells. The development of pulmonary eosinophilia in formalin-inactivated RSV-vaccinated mice required the presence of the G and/or SH protein in the challenge virus. These data show that G and/or SH protein has a marked impact on the inflammatory and innate immune response to RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"G and/or SH protein has a marked impact on the inflammatory and innate immune response to RSV infection and the development of pulmonary eosinophilia in formalin-inactivated RSV-vaccinated mice required the presence of the G and or SH protein in the challenge virus."}},"tag":"EXP"},{"id":326,"details":{"paperId":"6e1d00a7f4e5c6526b6ba6fb8b095beeec0c4fba","externalIds":{"MAG":"2099239352","DOI":"10.1128/JVI.06096-11","CorpusId":"25232618","PubMed":"22090124"},"title":"Respiratory Syncytial Virus Glycoprotein G Interacts with DC-SIGN and L-SIGN To Activate ERK1 and ERK2","abstract":"ABSTRACT Respiratory syncytial virus (RSV) interaction with epithelial and dendritic cells (DCs) is known to require divalent cations, suggesting involvement of C-type lectins. RSV infection and maturation of primary human DCs are reduced in a dose-dependent manner by EDTA. Therefore, we asked whether RSV infection involves DC-SIGN (CD209) or its isoform L-SIGN (CD299) (DC-SIGN/R). Using surface plasmon resonance analysis, we demonstrated that the attachment G glycoprotein of RSV binds both DC- and L-SIGN. However, neutralization of DC- and L-SIGN on primary human DCs did not inhibit RSV infection, demonstrating that interactions between RSV G and DC- or L-SIGN are not required for productive infection. Thus, neither DC- nor L-SIGN represents a functional receptor for RSV. However, inhibition of these interactions increased DC activation, as evidenced by significantly higher levels of alpha interferon (IFN-α), MIP-1α, and MIP-1β in plasmacytoid DCs (pDCs) exposed to RSV after neutralization of DC-and L-SIGN. To understand the molecular interactions involved, intracellular signaling events triggered by purified RSV G glycoprotein were examined in DC- and L-SIGN-transfected 3T3 cells. RSV G interaction with DC- or L-SIGN was shown to stimulate ERK1 and ERK2 phosphorylation, with statistically significant increases relative to mock-infected cells. Neutralization of DC- and L-SIGN reduced ERK1/2 phosphorylation. With increased DC activation following DC- and L-SIGN neutralization and RSV exposure, these data demonstrate that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Increased DC activation following DC- and L-SIGN neutralization and RSV exposure is demonstrated, demonstrating that the signaling events mediated by RSV G interactions with DC/L-SIGN are immunomodulatory and diminish DC activation, which may limit induction of RSV-specific immunity."}},"tag":"EXP"},{"id":507,"details":{"paperId":"4d80b553acaef6ce8812ab594b09ed990513caa3","externalIds":{"MAG":"1598802044","DOI":"10.4049/jimmunol.176.3.1733","CorpusId":"1487076","PubMed":"16424203"},"title":"Respiratory Syncytial Virus Induces TLR3 Protein and Protein Kinase R, Leading to Increased Double-Stranded RNA Responsiveness in Airway Epithelial Cells1","abstract":"Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-κB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner, which leads to increased NF-κB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA."}},"tag":"EXP"},{"id":144,"details":{"paperId":"0198a84f4159af235c61454b2f8b0035bb5df24a","externalIds":{"DOI":"10.1038/s41598-017-18672-5","CorpusId":"256947049"},"title":"Respiratory Syncytial Virus Infection Changes Cargo Composition of Exosome Released from Airway Epithelial Cells","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is observed that both RNA and protein signatures of RSV were present in exosomes, however, they were not able to establish productive infection in uninfected cells, suggesting understating their role in RSV infection may open new avenues for target identification and development of novel therapeutics."}},"tag":"EXP"},{"id":433,"details":{"paperId":"70fdbfbe022829996153aaf4610eed6241f2c94f","externalIds":{"PubMedCentral":"7720431","MAG":"3109756456","DOI":"10.12659/MSM.926763","CorpusId":"227252806","PubMed":"33262321"},"title":"Respiratory Syncytial Virus Infection Reduces Kynurenic Acid Production and Reverses Th17/Treg Balance by Modulating Indoleamine 2,3-Dioxygenase (IDO) Molecules in Plasmacytoid Dendritic Cells","abstract":"Background Respiratory syncytial virus (RSV) infection causes a world-wide medical and economic burden. This study analyzed the effects of RSV infection on plasmacytoid dendritic cells (pDCs) and evaluated the immunopathogenesis of RSV infection by measuring relative numbers of FoxP3+ Treg cells and Th17 cells. Material/Methods pDCs were isolated from human blood samples, purified using magnetic microbeads, and treated with RSV, IFN-γ, or vehicle. These cells were mixed with purified CD4+ T cells to yield preparations of pDCs+T cells+vehicle, pDCs+T cells+RSV, and pDCs+T cells+IFN-γ. Preparations of pDCs+T cells+RSV were also incubated with an inducer or an inhibitor of indoleamine 2,3-dioxygenase (IDO). Kynurenic acid concentration was measured by high-pressure liquid chromatography (HPLC). The differentiation of Foxp3+ Treg and Th17 cells from CD4+ T cells was determined by flow cytometry. Results pDCs were successfully isolated and purified using the magnetic microbeads. Compared with preparations of pDCs+T cells+vehicle, RSV infection (pDCs+T cells+RSV) significantly reduced and IFN-γ treatment (pDC+T cells+IFN-γ) increased kynurenic acid concentrations and the proportions of Foxp3+ Tregs (p<0.05 each). Conversely, RSV infection increased and IFN-γ treatment decreased the proportions of Th17 cells (p<0.05 each). RSV infection reduced kynurenic acid concentrations and inhibited the transformation from Th17 to Foxp3+ Tregs by modulating IDO molecules. Conclusions RSV infection reduced the production of kynurenic acid and inhibited transformation from Th17 to Foxp3+ Tregs (Th17/Treg balance) by modulating IDO molecules in pDCs.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV infection reduced the production of kynurenic acid and inhibited transformation from Th17 to Foxp3+ Tregs (Th17/Treg balance) by modulating IDO molecules in pDCs."}},"tag":"EXP"},{"id":279,"details":{"paperId":"f979db7d22e0b68a0df0eea2911e9f81bdc214c9","externalIds":{"MAG":"1564498973","DOI":"10.1128/JVI.00349-15","CorpusId":"24518067","PubMed":"25972545"},"title":"Respiratory Syncytial Virus Infection Upregulates NLRC5 and Major Histocompatibility Complex Class I Expression through RIG-I Induction in Airway Epithelial Cells","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid-inducible gene I (RIG-I). We show here that RSV infection upregulates major histocompatibility complex class I (MHC-I) expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via beta interferon (IFN-β) production, since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFN-β, but not by antibody to IFN-γ. RSV infection resulted in RIG-I upregulation and induction of NLRC5 and MHC-I. Suppression of RIG-I induction significantly blocked NLRC5, as well as MHC-I, upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC-I following introduction of the RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore proposed in which the host upregulates MHC-I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC-I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease. IMPORTANCE Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Infection early in life is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I (MHC-I) on the cell surface, which facilitates cytotoxic T cell activation and antiviral immunity. Here, we show that RSV infection of lung epithelial cells induces expression of RIG-I, resulting in induction of a class I MHC transactivator, NLRC5, and subsequent upregulation of MHC-I. Suppression of RIG-I induction blocked RSV-induced NLRC5 expression and MHC-I upregulation. Increased MHC-I expression may exacerbate the RSV disease condition due to immunopathologic damage, linking the innate immune response to RSV disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV infection of lung epithelial cells induces expression of RIG-I, resulting in induction of a class I MHC transactivator, NLRC5, and subsequent upregulation of MHC-I."}},"tag":"EXP"},{"id":373,"details":{"paperId":"94b5dd556afaeb337fc4a4157a0ad7628faaab63","externalIds":{"MAG":"2066243349","DOI":"10.1128/JVI.76.11.5654-5666.2002","CorpusId":"14343238","PubMed":"11991994"},"title":"Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology","abstract":"ABSTRACT Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Investigation of human primary airway epithelial cell cultures revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months."}},"tag":"EXP"},{"id":410,"details":{"paperId":"770dfac7df138c6de445d92f3873ba45eaf4515f","externalIds":{"PubMedCentral":"4095650","MAG":"2017381668","DOI":"10.1155/2014/850831","CorpusId":"10524453","PubMed":"25089282"},"title":"Respiratory Syncytial Virus Infections in Infants Affected by Primary Immunodeficiency","abstract":"Primary immunodeficiencies are rare inherited disorders that may lead to frequent and often severe acute respiratory infections. Respiratory syncytial virus (RSV) is one of the most frequent pathogens during early infancy and the infection is more severe in immunocompromised infants than in healthy infants, as a result of impaired T- and B-cell immune response unable to efficaciously neutralize viral replication, with subsequent increased viral shedding and potentially lethal lower respiratory tract infection. Several authors have reported a severe clinical course after RSV infections in infants and children with primary and acquired immunodeficiencies. Environmental prophylaxis is essential in order to reduce the infection during the epidemic season in hospitalized immunocompromised infants. Prophylaxis with palivizumab, a humanized monoclonal antibody against the RSV F protein, is currently recommended in high-risk infants born prematurely, with chronic lung disease or congenital heart disease. Currently however the prophylaxis is not routinely recommended in infants with primary immunodeficiency, although some authors propose the extension of prophylaxis to this high risk population.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Prophylaxis with palivizumab, a humanized monoclonal antibody against the RSV F protein, is currently recommended in high-risk infants born prematurely, with chronic lung disease or congenital heart disease, however the prophylactic is not routinely recommended in infants with primary immunodeficiency, although some authors propose the extension of prophYLaxis to this high risk population."}},"tag":"EXP"},{"id":199,"details":{"paperId":"5709f7e445d823292085b2dd043f0270d602b85f","externalIds":{"PubMedCentral":"6376914","MAG":"2893710367","DOI":"10.1093/infdis/jiy566","CorpusId":"52812829","PubMed":"30252097"},"title":"Respiratory Syncytial Virus Infects Primary Neonatal and Adult Natural Killer Cells and Affects Their Antiviral Effector Function","abstract":"Abstract Background Respiratory syncytial virus (RSV) is a major cause of severe acute lower respiratory tract infections in infants. Natural killer (NK) cells are important antiviral effector cells that likely encounter RSV in the presence of virus-specific (maternal) antibodies. As NK cells potentially contribute to immunopathology, we investigated whether RSV affects their antiviral effector functions. Methods We assessed the phenotype and functionality of primary neonatal and adult NK cells by flow cytometry after stimulation with RSV or RSV-antibody complexes. Results We demonstrate for the first time that RSV infects neonatal and adult NK cells in vitro. Preincubation of virus with subneutralizing concentrations of RSV-specific antibodies significantly increased the percentage of infected NK cells. Upon infection, NK cells were significantly more prone to produce interferon-γ, while secretion of the cytotoxicity molecule perforin was not enhanced. Conclusions Our findings suggest that (antibody-enhanced) RSV infection of NK cells induces a proinflammatory rather than a cytotoxic response, which may contribute to immunopathology. Considering that most RSV vaccines currently being developed aim at inducing (maternal) antibodies, these results highlight the importance of understanding the interactions between innate effector cells and virus-specific antibodies.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated for the first time that RSV infects neonatal and adult NK cells in vitro and the findings suggest that (antibody-enhanced) RSV infection of NK cells induces a proinflammatory rather than a cytotoxic response, which may contribute to immunopathology."}},"tag":"EXP"},{"id":65,"details":{"paperId":"802c4d31c478c807e19d0a832f84ec4eacefa0d6","externalIds":{"PubMedCentral":"7128247","MAG":"2589772520","DOI":"10.1016/j.immuni.2017.01.010","CorpusId":"191460","PubMed":"28228284"},"title":"Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A neonatal‐specific regulatory B cell is found that is recruited in the respiratory tract, gets infected by the virus, and correlates with high viral load and disease severity and is predictive of the severity of acute bronchiolitis disease."}},"tag":"EXP"},{"id":172,"details":{"paperId":"3e99f96cb0a38b483968671d02d29e80cb1e1b7c","externalIds":{"MAG":"2000818889","DOI":"10.1074/JBC.M108107200","CorpusId":"84694422"},"title":"Respiratory Syncytial Virus Inhibits Apoptosis and Induces NF-κB Activity through a Phosphatidylinositol 3-Kinase-dependent Pathway*","abstract":"Respiratory syncytial virus (RSV) infects airway epithelial cells, resulting in cell death and severe inflammation through the induction of NF-κB activity and inflammatory cytokine synthesis. Both NF-κB activity and apoptosis regulation have been linked to phosphatidylinositol 3-kinase (PI 3-K) and its downstream effector enzymes, AKT and GSK-3. This study evaluates the role of PI 3-K and its downstream mediators in apoptosis and inflammatory gene induction during RSV infection of airway epithelial cells. Whereas RSV infection alone did not produce significant cytotoxicity until 24–48 h following infection, simultaneous RSV infection and exposure to LY294002, a blocker of PI 3-K activity, resulted in cytotoxicity within 12 h. Furthermore, we found that RSV infection during PI 3-K blockade resulted in apoptosis by examining DNA fragmentation, DNA labeling by terminal dUTP nick-end labeling assay, and poly(ADP-ribose) polymerase cleavage by Western blotting. RSV infection produced an increase in the phosphorylation state of AKT, GSK-3, and the p85 regulatory subunit of PI 3-K. The activation of PI 3-K by RSV and its inhibition by LY294002 was confirmed in direct PI 3-K activity assays. Further evidence for the central role of a pathway involving PI 3-K and AKT in preserving cell viability during RSV infection was established by the observation that constitutively active AKT transfected into A549 cells prevented the cytotoxicity and apoptosis of combined RSV and LY294002 treatment. Finally, both PI 3-K inhibition by LY294002 and AKT inhibition by transfection of a dominant negative enzyme blocked RSV-induced NF-κB transcriptional activity. These data demonstrate that anti-apoptotic signaling and NF-κB activation by RSV are mediated through activation of PI 3-K-dependent pathways. Blockade of PI 3-K activation resulted in rapid, premature apoptosis and inhibition of RSV-stimulated NF-κB-dependent gene transcription.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is found that RSV infection during PI 3-K blockade resulted in apoptosis by examining DNA fragmentation, DNA labeling by terminal dUTP nick-end labeling assay, and poly(ADP-ribose) polymerase cleavage by Western blotting, and these data demonstrate that anti-apoptotic signaling and NF-κB activation by RSV are mediated through activation of PI3-K-dependent pathways."}},"tag":"EXP"},{"id":508,"details":{"paperId":"9f8c2956ca85a89231dbb5170924e6c01b84a156","externalIds":{"MAG":"1499834365","DOI":"10.4049/jimmunol.176.9.5529","CorpusId":"82159069"},"title":"Respiratory Syncytial Virus Inhibits Granulocyte Apoptosis through a Phosphatidylinositol 3-Kinase and NF-κB-Dependent Mechanism1","abstract":"Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-κB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intrACEllular TLRs in mediating these effects."}},"tag":"EXP"},{"id":179,"details":{"paperId":"b9924b220050bf80e5b54d133d55011020fb95cf","externalIds":{"MAG":"2014859464","DOI":"10.1074/jbc.M806816200","CorpusId":"35848110","PubMed":"19131335"},"title":"Respiratory Syncytial Virus Inhibits Lung Epithelial Na+ Channels by Up-regulating Inducible Nitric-oxide Synthase*","abstract":"Respiratory syncytial virus (RSV) infection has been shown to reduce Na+-driven alveolar fluid clearance in BALB/c mice in vivo. To investigate the cellular mechanisms by which RSV inhibits amiloride-sensitive epithelial Na+ channels (ENaC), the main pathways through which Na+ ions enter lung epithelial cells, we infected human Clara-like lung (H441) cells with RSV that expresses green fluorescent protein (rRA2). 3-6 days later patch clamp recordings showed that infected cells (i.e. cells expressing green fluorescence; GFP(+)) had significantly lower whole-cell amiloride-sensitive currents and single channel activity (NPo) as compared with non-infected (GFP(-)), non-inoculated, or cells infected with UV-inactivated RSV. Both α and β ENaC mRNA levels were significantly reduced in GFP(+) cells as measured by real-time reverse transcription-PCR. Infection with RSV increased expression of the inducible nitric-oxide synthase (iNOS) and nitrite concentration in the culture medium; nuclear translocation of NF-κB p65 subunit and NF-κB activation were also up-regulated. iNOS up-regulation in GFP(+) cells was prevented by knocking down IκB kinase γ before infection. Furthermore, pretreatment of H441 cells with the specific iNOS inhibitor 1400W (1 μm) resulted in a doubling of the amiloride-sensitive Na+ current in GFP(+) cells. Additionally, preincubation of H441 cells with A77-1726 (20 μm), a de novo UTP synthesis inhibitor, and 1400W completely reversed the RSV inhibition of amiloride-sensitive currents in GFP(+) cells. Thus, both UTP- and iNOS-generated reactive species contribute to ENaC down-regulation in RSV-infected airway epithelial cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Both UTP- and iNOS-generated reactive species contribute to ENaC down-regulation in RSV-infected airway epithelial cells, according to real-time reverse transcription-PCR."}},"tag":"EXP"},{"id":443,"details":{"paperId":"3352da8fea318282ac52a7f91579d68aefbdbc65","externalIds":{"PubMedCentral":"3080852","MAG":"2063875739","DOI":"10.1371/journal.ppat.1001336","CorpusId":"14101984","PubMed":"21533073"},"title":"Respiratory Syncytial Virus Interferon Antagonist NS1 Protein Suppresses and Skews the Human T Lymphocyte Response","abstract":"We recently demonstrated that the respiratory syncytial virus (RSV) NS1 protein, an antagonist of host type I interferon (IFN-I) production and signaling, has a suppressive effect on the maturation of human dendritic cells (DC) that was only partly dependent on released IFN-I. Here we investigated whether NS1 affects the ability of DC to activate CD8+ and CD4+ T cells. Human DC were infected with RSV deletion mutants lacking the NS1 and/or NS2 genes and assayed for the ability to activate autologous T cells in vitro, which were analyzed by multi-color flow cytometry. Deletion of the NS1, but not NS2, protein resulted in three major effects: (i) an increased activation and proliferation of CD8+ T cells that express CD103, a tissue homing integrin that directs CD8+ T cells to mucosal epithelial cells of the respiratory tract and triggers cytolytic activity; (ii) an increased activation and proliferation of Th17 cells, which have recently been shown to have anti-viral effects and also indirectly attract neutrophils; and (iii) decreased activation of IL-4-producing CD4+ T cells - which are associated with enhanced RSV disease - and reduced proliferation of total CD4+ T cells. Except for total CD4+ T cell proliferation, none of the T cell effects appeared to be due to increased IFN-I signaling. In the infected DC, deletion of the NS1 and NS2 genes strongly up-regulated the expression of cytokines and other molecules involved in DC maturation. This was partly IFN-I-independent, and thus might account for the T cell effects. Taken together, these data demonstrate that the NS1 protein suppresses proliferation and activation of two of the protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes proliferation and activation of Th2 cells that can enhance RSV disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data demonstrate that the NS1 protein suppresses proliferation and activation of two of the protective cell populations (CD103+ CD8+ T cells and Th17 cells), and promotes proliferation andactivation of Th2 cells that can enhance RSV disease."}},"tag":"EXP"},{"id":173,"details":{"paperId":"476e1d847af2402915b68553c6df08b0dbe86055","externalIds":{"MAG":"2143703838","DOI":"10.1074/jbc.M109.077321","CorpusId":"28394407","PubMed":"20519500"},"title":"Respiratory Syncytial Virus Limits α Subunit of Eukaryotic Translation Initiation Factor 2 (eIF2α) Phosphorylation to Maintain Translation and Viral Replication*","abstract":"The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR), a cellular kinase relevant to limiting viral replication (Groskreutz, D. J., Monick, M. M., Powers, L. S., Yarovinsky, T. O., Look, D. C., and Hunninghake, G. W. (2006) J. Immunol. 176, 1733–1740). It is activated by autophosphorylation, likely triggered by a double-stranded RNA intermediate during replication of the virus. In most instances, ph-PKR targets the α subunit of eukaryotic translation initiation factor 2 (eIF2α) protein via phosphorylation, leading to an inhibition of translation of cellular and viral protein. However, we found that although ph-PKR increases in RSV infection, significant eIF2α phosphorylation is not observed, and inhibition of protein translation does not occur. RSV infection attenuates eIF2α phosphorylation by favoring phosphatase rather than kinase activity. Although PKR is activated, RSV sequesters PKR away from eIF2α by binding of the kinase to the RSV N protein. This occurs in conjunction with an increase in the association of the phosphatase, PP2A, with eIF2α following PKR activation. The result is limited phosphorylation of eIF2α and continued translation of cellular and viral proteins.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that although ph-PKR increases in RSV infection, significant eIF2α phosphorylation is not observed, and inhibition of protein translation does not occur."}},"tag":"EXP"},{"id":372,"details":{"paperId":"44b3e2720e0be548761717f2579eb998c2f7b521","externalIds":{"MAG":"2159783312","DOI":"10.1128/JVI.75.24.12188-12197.2001","CorpusId":"21043063","PubMed":"11711610"},"title":"Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection","abstract":"ABSTRACT The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in Escherichia coli could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1–N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data show that phosphorylation is important for the M2-1 protein function in transcription, however, mutation of the M1-1 phosphorylated sites did not interfere with the ability of the H2O protein to interact with the N protein in transfected cells."}},"tag":"EXP"},{"id":497,"details":{"paperId":"5071744ce44946e1f82f842e71c57676ec39b3ca","externalIds":{"PubMedCentral":"6213044","MAG":"2894614613","DOI":"10.3390/v10100535","CorpusId":"52896645","PubMed":"30274351"},"title":"Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture","abstract":"The virus–host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1–256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for M–actin interaction in transporting virion components in the infected cell.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A possible role for M–actin interaction in transporting virion components in the infected cell is suggested, using deletion mutants of M in a transfected cell system, where both the N- and C-terminus of the protein are required for the interaction."}},"tag":"EXP"},{"id":435,"details":{"paperId":"9de12f22f036779afddbce4a66d16746f700b72a","externalIds":{"PubMedCentral":"3360651","MAG":"1968339049","DOI":"10.1371/journal.pone.0038052","CorpusId":"6335897","PubMed":"22662266"},"title":"Respiratory Syncytial Virus Matrix Protein Induces Lung Epithelial Cell Cycle Arrest through a p53 Dependent Pathway","abstract":"Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication."}},"tag":"EXP"},{"id":487,"details":{"paperId":"d8e5252ca3f599db8934e8cdf9e606445e604030","externalIds":{"PubMedCentral":"8534903","DOI":"10.3390/cells10102786","CorpusId":"239468091","PubMed":"34685766"},"title":"Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells","abstract":"The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M’s RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that M, dependent on amino acids 110–183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition."}},"tag":"EXP"},{"id":434,"details":{"paperId":"039f6d6a277237c986578fdc1e2cbcf1a4ee2901","externalIds":{"MAG":"2028571879","PubMedCentral":"3288005","DOI":"10.1371/journal.pone.0029386","CorpusId":"1172871","PubMed":"22383950"},"title":"Respiratory Syncytial Virus NS1 Protein Colocalizes with Mitochondrial Antiviral Signaling Protein MAVS following Infection","abstract":"Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production."}},"tag":"EXP"},{"id":414,"details":{"paperId":"41ffc3c7fc24d82cde491445d9a27c932dc1f96f","externalIds":{"MAG":"1982927008","DOI":"10.1159/000357327","CorpusId":"16945741","PubMed":"24480984"},"title":"Respiratory Syncytial Virus NS1 Protein Degrades STAT2 by Inducing SOCS1 Expression","abstract":"Objectives: Respiratory syncytial virus (RSV) nonstructural protein NS1 (NS1) has been shown to block interferon (IFN)-inducible antiviral signaling. The suppressor of cytokine signaling (SOCS) gene family could utilize a feedback loop to block the activation of the JAK/STAT signaling pathway, further inhibiting the activation of host type I IFN. We evaluated the role of the SOCS1 and SOCS3 genes in this antiviral mechanism. Material and Methods: A humanized stable NS1 (rich in GC)-expressing plasmid was constructed, and A549 cells were transfected with it. Expression of the SOCS1, SOCS3, RIG-I, and TLR3 mRNAs was measured with real-time PCR. STAT2 and pSTAT2 expression was determined with Western blotting. Results: RSV NS1 upregulated SOCS1 mRNA expression 30-fold increase compared with the baseline level in very early phase (p < 0.01), and silence of RIG-I or TLR3 mRNA did not affect NS1-induced SOCS1 expression. NS1 inhibited IFN-α-induced STAT2 phosphorylation and degraded STAT2 in a time-dependent manner compared with the empty-vector control (p < 0.05). Conclusion: RSV NS1 upregulates SOCS1 expression in a RIG-I- and TLR3-independent pathway, to inhibit STAT2 phosphorylation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV NS1 upregulates SOCS1 expression in a RIG-I- and TLR3-independent pathway, to inhibit STAT2 phosphorylation and degraded STAT2 in a time-dependent manner compared with the empty-vector control."}},"tag":"EXP"},{"id":313,"details":{"paperId":"7af8681c6d68cc3cbeffafc9001d3dd90cccc537","externalIds":{"MAG":"1966164449","DOI":"10.1128/JVI.02303-06","CorpusId":"30410104","PubMed":"17251292"},"title":"Respiratory Syncytial Virus NS1 Protein Degrades STAT2 by Using the Elongin-Cullin E3 Ligase","abstract":"ABSTRACT Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Investigating whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome indicates that E3 ligase activity is crucial for the ability of RSV to degrade STAT2."}},"tag":"EXP"},{"id":198,"details":{"paperId":"17fd9db0416f91d678a386850f228be07be10717","externalIds":{"MAG":"2773246759","DOI":"10.1093/infdis/jix445","CorpusId":"6565529","PubMed":"28968829"},"title":"Respiratory Syncytial Virus Nonstructural Protein 1 Blocks Glucocorticoid Receptor Nuclear Translocation by Targeting IPO13 and May Account for Glucocorticoid Insensitivity","abstract":"Despite their powerful antiinflammatory effect, glucocorticoids have shown no significant clinical benefit in respiratory syncytial virus (RSV)-induced bronchiolitis, the reason for which remains unclear. Upon glucocorticoid binding, the cytoplasmic glucocorticoid receptor (GR) translocates to the nucleus with the help of importin 13 (IPO13). Here, we report that RSV infection reduced GR nuclear translocation in nasopharyngeal aspirates from RSV-infected infants, lungs of infected mice, and A549 cells, which coincided with decreased IPO13 expression. This led to repression of GR-induced antiinflammatory genes, such that dexamethasone failed to suppress airway inflammation and airway hyperresponsiveness in the infected mice. The anti-GR effect of RSV was mediated by viral nonstructural protein 1 , which likely functioned by competing with IPO13 for GR binding. Our findings provide a mechanism for the ineffectiveness of glucocorticoids in RSV-related disease and highlight the potential to target the IPO13-GR axis as a treatment for multiple glucocorticoid-related diseases.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is reported that RSV infection reduced GR nuclear translocation in nasopharyngeal aspirates from RSV-infected infants, lungs of infected mice, and A549 cells, which coincided with decreased IPO13 expression, which led to repression of GR-induced antiinflammatory genes, such that dexamethasone failed to suppress airway inflammation and airway hyperresponsiveness in the infected mice."}},"tag":"EXP"},{"id":67,"details":{"paperId":"9ddc66d711f1ce4069f8bbf010fc0fc252ed78ba","externalIds":{"MAG":"2945900726","DOI":"10.1016/j.jaci.2019.05.014","CorpusId":"165163287","PubMed":"31128120"},"title":"Respiratory Syncytial Virus Nonstructural Protein 1 Downregulates Glucocorticoid Receptor Expression via miR-29a.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Infants with bronchiolitis do not respond to steroids, and glucocorticoid receptor (GR) is downregulated in RSV infected airway epithelial cells (AECs) and it is shown that RSVNS1 downregulates GR expression in amiR-29adependent manner."}},"tag":"EXP"},{"id":411,"details":{"paperId":"427a03969b078093df66df5f4ff5b9c9543abc18","externalIds":{"PubMedCentral":"9146443","DOI":"10.1155/2022/4086710","CorpusId":"248973728","PubMed":"35637792"},"title":"Respiratory Syncytial Virus Nonstructural Protein 1 Promotes 5-Lipoxygenase via miR-19a-3p","abstract":"Background Respiratory syncytial virus (RSV) infection can regulate the expression of a wide range of noncoding microRNAs (miRNAs), in which mir-19a-3p can participate in airway inflammatory response by regulating 5-lipoxygenase (5-LO) pathway. RSV nonstructural protein (NS) 1 is involved in the airway hyperresponsiveness during RSV infection. Methods The expression levels of miR-19a-3p and inflammatory signaling-related indicators were detected using quantitative real-time PCR and western blot analyses on the A549 cells transfected with NS1 expression plasmids (pNS1). The 5-LO-mediated inflammatory signaling pathway was assessed when the miR-19a-3p or 5-LO was inhibited. Results The immunofluorescence analysis showed that the plasmid-mediated NS1 protein was observed in both the cytoplasm and nucleus. The expression level of miR-19a-3p was significantly upregulated in the pNS1 or RSV-treated cells, which was reversed by the NS1 small interfering RNA. In addition, pNS1 also upregulated the expression of 5-LO, interleukin-5 (IL-5), and leukotriene B4 (LTB4), which was also significantly inhibited by the miR-19a-3p antagonists. The 5-LO inhibitor MK886 prevented the increase in the expression level of IL-5 induced by pNS1. Conclusions These results suggested that the RSV NS1 might play an important role in the pathogenesis of RSV by activating the 5-LO and subsequent inflammatory cytokines through miR-19a-3p.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results suggested that the RSV NS1 might play an important role in the pathogenesis of RSV by activating the 5-LO and subsequent inflammatory cytokines through miR-19a-3p."}},"tag":"EXP"},{"id":389,"details":{"paperId":"e284273e0d829fabb474ebd62b472d32830a95b8","externalIds":{"MAG":"2051874642","DOI":"10.1128/JVI.79.14.9315-9319.2005","CorpusId":"11969916","PubMed":"15994826"},"title":"Respiratory Syncytial Virus Nonstructural Proteins NS1 and NS2 Mediate Inhibition of Stat2 Expression and Alpha/Beta Interferon Responsiveness","abstract":"ABSTRACT Respiratory syncytial virus (RSV) subverts the antiviral interferon (IFN) response, but the mechanism for this evasion was unclear. Here we show that RSV preferentially inhibits IFN-α/β signaling by expression of viral NS1 and NS2. Thus, RSV infection or expression of recombinant NS1 and NS2 in epithelial host cells causes a marked decrease in Stat2 levels and the consequent downstream IFN-α/β response. Similarly, NS1/NS2-deficient RSV no longer decreases Stat2 levels or IFN responsiveness. RSV infection decreased human but not mouse Stat2 levels, so this mechanism of IFN antagonism may contribute to viral host range, as well as immune subversion.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV preferentially inhibits IFN-α/β signaling by expression of viral NS1 and NS2 in epithelial host cells, which may contribute to viral host range, as well as immune subversion."}},"tag":"EXP"},{"id":295,"details":{"paperId":"fb64c315f579cf08c4e2c94247d13dd5bec5d8e1","externalIds":{"DOI":"10.1128/JVI.01206-21","CorpusId":"238422256","PubMed":"34613802"},"title":"Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication","abstract":"RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen. ABSTRACT Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis."}},"tag":"EXP"},{"id":299,"details":{"paperId":"5bf822b1741626b784e6fabeb5a704968e038dc8","externalIds":{"MAG":"3083142408","PubMedCentral":"7592213","DOI":"10.1128/JVI.01380-20","CorpusId":"221475618","PubMed":"32878896"},"title":"Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling","abstract":"Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease. ABSTRACT Viruses routinely employ strategies to prevent the activation of innate immune signaling in infected cells. Respiratory syncytial virus (RSV) is no exception, as it encodes two accessory proteins (NS1 and NS2) which are well established to block interferon signaling. However, RSV-encoded mechanisms for inhibiting NF-κB signaling are less well characterized. In this study, we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV-infected cells, we demonstrated that the p65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured p65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following tumor necrosis factor alpha (TNF-α) stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalize the RSV N protein and p65 within bovine RSV (bRSV) IBs, which are granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterization of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signaling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate—a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally, they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells. IMPORTANCE Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This study identifies a novel mechanism for viral antagonism of innate immune signaling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate—a mechanism conserved across the Orthopneumovirus genus and not host-cell specific."}},"tag":"EXP"},{"id":448,"details":{"paperId":"e88797458f110553b9418007d35401975dce1454","externalIds":{"PubMedCentral":"4676609","MAG":"2198743132","DOI":"10.1371/journal.ppat.1005318","CorpusId":"8924581","PubMed":"26658574"},"title":"Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures","abstract":"Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization."}},"tag":"EXP"},{"id":134,"details":{"paperId":"56e0de5b9dd437c209dfc97e960de0b8863235f9","externalIds":{"MAG":"798319868","DOI":"10.1038/mt.2015.124","CorpusId":"38227047","PubMed":"26156244"},"title":"Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This report represents the first identification of a natural target of a tRF and illustrates how a virus utilizes a host tRF to control a host gene to favor its replication."}},"tag":"EXP"},{"id":145,"details":{"paperId":"ba1bf566a85367144193d8064272229f348d3dc6","externalIds":{"DOI":"10.1038/s41598-018-32576-y","CorpusId":"256952520"},"title":"Respiratory Syncytial Virus induces the classical ROS-dependent NETosis through PAD-4 and necroptosis pathways activation","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is found that RSV infection of alveolar epithelial cells or lung fibroblasts triggers NET-DNA release by neutrophil, indicating that neutrophils can identify RSV-infected cells and respond to them by releasing NETs."}},"tag":"EXP"},{"id":477,"details":{"paperId":"ba400fb441fcadc30cb8222b4e50c10820ea03fa","externalIds":{"PubMedCentral":"7248305","MAG":"3027368528","DOI":"10.3389/fcimb.2020.00225","CorpusId":"218676157","PubMed":"32509597"},"title":"Respiratory Syncytial Virus's Non-structural Proteins: Masters of Interference","abstract":"Respiratory Syncytial Virus (RSV) is a highly prevalent virus that affects the majority of the population. The virus can cause severe disease in vulnerable populations leading to high hospitalization rates from bronchiolitis or secondary bacterial infections leading to pneumonia. Two early and non-structural proteins (Ns1 and Ns2), strongly over-ride the antiviral innate system but also diminish the adaptive response as well. This review will cover interactions of Ns1 and Ns2 with the host antiviral response with a focus on alterations to signaling pathways, cytokine gene expression, and effects of the Ns proteins on mitochondria.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This review will cover interactions of Ns1 and Ns2 with the host antiviral response with a focus on alterations to signaling pathways, cytokine gene expression, and effects of the Ns proteins on mitochondria."}},"tag":"EXP"},{"id":272,"details":{"paperId":"d3f7245db553c66e233732d0600e18d03c2c4691","externalIds":{"MAG":"2123896260","DOI":"10.1128/JVI.00142-10","CorpusId":"23161954","PubMed":"20410276"},"title":"Respiratory Syncytial Virus-Mediated NF-κB p65 Phosphorylation at Serine 536 Is Dependent on RIG-I, TRAF6, and IKKβ","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is the etiological agent of acute respiratory diseases, such as bronchiolitis and pneumonia. The exacerbated production of proinflammatory cytokines and chemokines in the airways in response to RSV is an important pillar in the development of these pathologies. As such, a keen understanding of the mechanisms that modulate the inflammatory response during RSV infection is of pivotal importance to developing effective treatment. The NF-κB transcription factor is a major regulator of proinflammatory cytokine and chemokine genes. However, RSV-mediated activation of NF-κB is far from characterized. We recently demonstrated that aside from the well-characterized IκBα phosphorylation and degradation, the phosphorylation of p65 at Ser536 is an essential event regulating the RSV-mediated NF-κB-dependent promoter transactivation. In the present study, using small interfering RNA and pharmacological inhibitors, we now demonstrate that RSV sensing by the RIG-I cytoplasmic receptor triggers a signaling cascade involving the MAVS and TRAF6 adaptors that ultimately leads to p65ser536 phosphorylation by the IKKβ kinase. In a previous study, we highlighted a critical role of the NOX2-containing NADPH oxidase enzyme as an upstream regulator of both the IκBαSer32 and p65Ser536 in human airway epithelial cells. Here, we demonstrate that inhibition of NOX2 significantly decreases IKKβ activation. Taken together, our data identify a new RIG-I/MAVS/TRAF6/IKKβ/p65Ser536 pathway placed under the control of NOX2, thus characterizing a novel regulatory pathway involved in NF-κB-driven proinflammatory response in the context of RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that RSV sensing by the RIG-I cytoplasmic receptor triggers a signaling cascade involving the MAVS and TRAF6 adaptors that ultimately leads to p65ser536 phosphorylation by the IKKβ kinase, thus characterizing a novel regulatory pathway involved in NF-κB-driven proinflammatory response in the context of RSV infection."}},"tag":"EXP"},{"id":297,"details":{"paperId":"5da3e64a4cdd42bf37f365759059eac1b90f688f","externalIds":{"MAG":"2767754527","DOI":"10.1128/JVI.01302-17","CorpusId":"4867300","PubMed":"29118126"},"title":"Respiratory Syncytial Virus: Targeting the G Protein Provides a New Approach for an Old Problem","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) annually affecting >2 million children in the United States <5 years old. In the elderly (>65 years old), RSV results in ∼175,000 hospitalizations annually in the United States with a worldwide incidence of ∼34 million. There is no approved RSV vaccine, and treatments are limited. Recently, a phase 3 trial in the elderly using a recombinant RSV F protein vaccine failed to meet its efficacy objectives, namely, prevention of moderate-to-severe RSV-associated LRTI and reduced incidence of acute respiratory disease. Moreover, a recent phase 3 trial evaluating suptavumab (REGN2222), an antibody to RSV F protein, did not meet its primary endpoint of preventing medically attended RSV infections in preterm infants. Despite these setbacks, numerous efforts targeting the RSV F protein with vaccines, antibodies, and small molecules continue based on the commercial success of a monoclonal antibody (MAb) against the RSV F protein (palivizumab). As the understanding of RSV biology has improved, the other major coat protein, the RSV G protein, has reemerged as an alternative target reflecting progress in understanding its roles in infecting bronchial epithelial cells and in altering the host immune response. In mouse models, a high-affinity, strain-independent human MAb to the RSV G protein has shown potent direct antiviral activity combined with the alleviation of virus-induced immune system effects that contribute to disease pathology. This MAb, being prepared for clinical trials, provides a qualitatively new approach to managing RSV for populations not eligible for prophylaxis with palivizumab.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This MAb, being prepared for clinical trials, provides a qualitatively new approach to managing RSV for populations not eligible for prophylaxis with palivizumab."}},"tag":"EXP"},{"id":489,"details":{"paperId":"fa2e0e3d6002f780b7a478024aaf2e0a5d7cfa32","externalIds":{"PubMedCentral":"5578107","MAG":"2744019602","DOI":"10.3390/ijms18081717","CorpusId":"13775851","PubMed":"28783078"},"title":"Respiratory Syncytial Virus: The Influence of Serotype and Genotype Variability on Clinical Course of Infection","abstract":"Respiratory syncytial virus (RSV) belongs to the recently defined Pneumoviridae family, Orthopneumovirus genus. It is the leading cause of acute bronchiolitis and one of the most common causes of infant viral death worldwide, with infection typically occurring as recurrent seasonal epidemics. There are two major RSV subtypes, A and B, and multiple genotypes, which can coexist during RSV epidemic season every year and result in different disease severity. Recently, new RSV genomic sequences and analysis of RSV genotypes have provided important data for understanding RSV pathogenesis. Novel RSV strains do spread rapidly and widely, and a knowledge of viral strain-specific phenotypes may be important in order to include the more virulent strains in future therapeutical options and vaccine development. Here we summarize recent literature exploring genetic and molecular aspects related to RSV infection, their impact on the clinical course of the disease and their potential utility in the development of safe and effective preventive and therapeutic strategies.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Recent literature exploring genetic and molecular aspects related to RSV infection, their impact on the clinical course of the disease and their potential utility in the development of safe and effective preventive and therapeutic strategies are summarized."}},"tag":"OTHER"},{"id":169,"details":{"paperId":"7cfba35ec8d8deb261d9849bd7b38a84ddedfa52","externalIds":{"MAG":"2159482780","DOI":"10.1073/PNAS.94.25.13961","CorpusId":"8909740","PubMed":"9391135"},"title":"Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant.","abstract":"A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription-PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins, which suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate."}},"tag":"EXP"},{"id":41,"details":{"paperId":"eb488a5976bef681965cd8b62673dbbaddffc34f","externalIds":{"MAG":"1528331455","DOI":"10.1007/s11262-006-0064-x","CorpusId":"26218223","PubMed":"16972040"},"title":"Respiratory syncytial virus (RSV) evades the human adaptive immune system by skewing the Th1/Th2 cytokine balance toward increased levels of Th2 cytokines and IgE, markers of allergy—a review","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The aim of the present review is to base RSV pathogenicity on the numerous very good analyses of the virus genes and to suggest a therapeutic approach to treatment directed at preventing the inhibitory effects of Th2 cytokines on the adaptive immune system of the patients, instead of inhibiting RSV replication by antivirals."}},"tag":"EXP"},{"id":62,"details":{"paperId":"44f83b71e82570875e937ff1b6d57dcec10d58c8","externalIds":{"MAG":"2094601640","PubMedCentral":"3741917","DOI":"10.1016/j.fob.2013.07.005","CorpusId":"653770","PubMed":"23951552"},"title":"Respiratory syncytial virus (RSV) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation☆","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that the N‐terminal region of GR is required for RSV repression of GR transactivation and that RSV infection of lung epithelial cells reduces ligand‐dependent GR phosphorylation at serine 211 and serine 226."}},"tag":"EXP"},{"id":191,"details":{"paperId":"68595a5a4362baee2687d844b9210b57a9bf93d8","externalIds":{"MAG":"2001173923","DOI":"10.1086/651431","CorpusId":"205994264","PubMed":"20205592"},"title":"Respiratory syncytial virus F and G proteins induce interleukin 1alpha, CC, and CXC chemokine responses by normal human bronchoepithelial cells.","abstract":"Human respiratory syncytial virus (RSV) is a ubiquitous respiratory virus that causes serious lower respiratory tract disease in infants and young children worldwide. Studies have shown that RSV infection modulates chemokine expression patterns, suggesting that particular cytokine expression profiles may be indicators of disease severity. In this study, we show that RSV F or G protein treatment of fully differentiated primary normal human bronchial epithelial cells induces apical and basolateral secretion of interleukin 8 (IL-8), interferon-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), and RANTES (regulated on activation, normal T cell expressed and secreted). Purified RSV G (attachment) protein was shown to stimulate the secretion of interleukin 1alpha and RANTES, whereas purified F (fusion) protein elicited the production of IL-8, IP-10, and RANTES. Studies of ultraviolet-inactivated RSV showed that treatment of normal human bronchial epithelial cells induces apical IL-8, IP-10, and MCP-1 secretion independent of infection, suggesting that RSV proteins alone modify the chemokine response pattern, which may affect the early immune response before infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV F or G protein treatment of fully differentiated primary normal human bronchial epithelial cells induces apical and basolateral secretion of interleukin 8 (IL-8), interferon-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), and RANTES."}},"tag":"EXP"},{"id":464,"details":{"paperId":"0d41881af7bf7ffe58c85b1aca37666a59a2e171","externalIds":{"PubMedCentral":"8162694","DOI":"10.1371/journal.ppat.1009589","CorpusId":"234781624","PubMed":"34003848"},"title":"Respiratory syncytial virus M2-1 protein associates non-specifically with viral messenger RNA and with specific cellular messenger RNA transcripts","abstract":"Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants and the elderly. RSV is a non-segmented negative strand RNA virus. The viral M2-1 protein plays a key role in viral transcription, serving as an elongation factor to enable synthesis of full-length mRNAs. M2-1 contains an unusual CCCH zinc-finger motif that is conserved in the related human metapneumovirus M2-1 protein and filovirus VP30 proteins. Previous biochemical studies have suggested that RSV M2-1 might bind to specific virus RNA sequences, such as the transcription gene end signals or poly A tails, but there was no clear consensus on what RSV sequences it binds. To determine if M2-1 binds to specific RSV RNA sequences during infection, we mapped points of M2-1:RNA interactions in RSV-infected cells at 8 and 18 hours post infection using crosslinking immunoprecipitation with RNA sequencing (CLIP-Seq). This analysis revealed that M2-1 interacts specifically with positive sense RSV RNA, but not negative sense genome RNA. It also showed that M2-1 makes contacts along the length of each viral mRNA, indicating that M2-1 functions as a component of the transcriptase complex, transiently associating with nascent mRNA being extruded from the polymerase. In addition, we found that M2-1 binds specific cellular mRNAs. In contrast to the situation with RSV mRNA, M2-1 binds discrete sites within cellular mRNAs, with a preference for A/U rich sequences. These results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results suggest that in addition to its previously described role in transcription elongation, M2-1 might have an additional role involving cellular RNA interactions."}},"tag":"EXP"},{"id":79,"details":{"paperId":"ce8f1ccbaa5b04d2e17b96ddfede872e9022fb48","externalIds":{"MAG":"2026527418","DOI":"10.1016/J.VIROL.2004.10.031","CorpusId":"2276904","PubMed":"15629770"},"title":"Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-kappaB in A549 cells."}},"tag":"EXP"},{"id":514,"details":{"paperId":"092ac635ed5399a265516f79c2eeb68587706e03","externalIds":{"MAG":"2955030790","PubMedCentral":"6598784","DOI":"10.7554/eLife.42448","CorpusId":"195695478","PubMed":"31246170"},"title":"Respiratory syncytial virus co-opts host mitochondrial function to favour infectious virus production","abstract":"Although respiratory syncytial virus (RSV) is responsible for more human deaths each year than influenza, its pathogenic mechanisms are poorly understood. Here high-resolution quantitative imaging, bioenergetics measurements and mitochondrial membrane potential- and redox-sensitive dyes are used to define RSV’s impact on host mitochondria for the first time, delineating RSV-induced microtubule/dynein-dependent mitochondrial perinuclear clustering, and translocation towards the microtubule-organizing centre. These changes are concomitant with impaired mitochondrial respiration, loss of mitochondrial membrane potential and increased production of mitochondrial reactive oxygen species (ROS). Strikingly, agents that target microtubule integrity the dynein motor protein, or inhibit mitochondrial ROS production strongly suppresses RSV virus production, including in a mouse model with concomitantly reduced virus-induced lung inflammation. The results establish RSV’s unique ability to co-opt host cell mitochondria to facilitate viral infection, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV’s unique ability to co-opt host cell mitochondria to facilitate viral infection is established, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention."}},"tag":"OTHER"},{"id":77,"details":{"paperId":"a3a03c087230744e83d1e63585ab977fd22b588c","externalIds":{"MAG":"2013645823","DOI":"10.1016/J.VIROL.2004.10.004","CorpusId":"40041605","PubMed":"15567433"},"title":"Respiratory syncytial virus deficient in soluble G protein induced an increased proinflammatory response in human lung epithelial cells.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that RSV, in the course of an ongoing infection, reduces by the production of sG protein the detrimental inflammatory response evolved by the infected resident lung epithelial cell and thereby supports its own replication."}},"tag":"EXP"},{"id":142,"details":{"paperId":"bc5ea849d39e9fd6e298e785868afbd481e8648d","externalIds":{"DOI":"10.1038/s41579-019-0149-x","CorpusId":"256744981"},"title":"Respiratory syncytial virus entry and how to block it","abstract":null,"publicationTypes":["Review"],"tldr":{"model":"tldr@v2.0.0","text":"The recent findings that have begun to elucidate RSV entry mechanisms are summarized, progress on the development of new interventions are described and a perspective on gaps in knowledge that require further investigation is concluded."}},"tag":"EXP"},{"id":201,"details":{"paperId":"f1e54e111574fcbd5c10088cd2a761ad9beea07b","externalIds":{"MAG":"2027789488","DOI":"10.1093/NAR/13.5.1559","CorpusId":"1510512","PubMed":"2987829"},"title":"Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.","abstract":"The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing to identify a sequence of lys-lys-arg-lys -arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain."}},"tag":"EXP"},{"id":422,"details":{"paperId":"d946211ef6e1f3899b78b442f436b0bc2902866e","externalIds":{"PubMedCentral":"2584630","MAG":"2130087463","DOI":"10.1186/1743-422X-5-127","CorpusId":"1564843","PubMed":"18950517"},"title":"Respiratory syncytial virus glycoproteins uptake occurs through clathrin-mediated endocytosis in a human epithelial cell line","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that clathrin-mediated endocytosis is the major uptake route of RSV antigens by an epithelial human cell line."}},"tag":"EXP"},{"id":156,"details":{"paperId":"f14c1def8471e38c7e4aa730c4a7d0f14cb0ecd6","externalIds":{"MAG":"1984328509","DOI":"10.1073/pnas.0802555105","CorpusId":"21864521","PubMed":"18818306"},"title":"Respiratory syncytial virus impairs T cell activation by preventing synapse assembly with dendritic cells","abstract":"Respiratory syncytial virus (RSV) infection is one of the leading causes of infant hospitalization and a major health and economic burden worldwide. Infection with this virus induces an exacerbated innate proinflammatory immune response characterized by abundant immune cell infiltration into the airways and lung tissue damage. RSV also impairs the induction of an adequate adaptive T cell immune response, which favors virus pathogenesis. Unfortunately, to date there are no efficient vaccines against this virus. Recent in vitro and in vivo studies suggest that RSV infection can prevent T cell activation, a phenomenon attributed in part to cytokines and chemokines secreted by RSV-infected cells. Efficient immunity against viruses is promoted by dendritic cells (DCs), professional antigen-presenting cells, that prime antigen-specific helper and cytotoxic T cells. Therefore, it would be to the advantage of RSV to impair DC function and prevent the induction of T cell immunity. Here, we show that, although RSV infection induces maturation of murine DCs, these cells are rendered unable to activate antigen-specific T cells. Inhibition of T cell activation by RSV was observed independently of the type of TCR ligand on the DC surface and applied to cognate-, allo-, and superantigen stimulation. As a result of exposure to RSV-infected DCs, T cells became unresponsive to subsequent TCR engagement. RSV-mediated impairment in T cell activation required DC-T cell contact and involved inhibition of immunological synapse assembly among these cells. Our data suggest that impairment of immunological synapse could contribute to RSV pathogenesis by evading adaptive immunity and reducing T cell-mediated virus clearance.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that, although RSV infection induces maturation of murine DCs, these cells are rendered unable to activate antigen-specific T cells, suggesting that impairment of immunological synapse could contribute to RSV pathogenesis by evading adaptive immunity and reducing T cell-mediated virus clearance."}},"tag":"EXP"},{"id":64,"details":{"paperId":"f9f821bd288e7ab19aea4ae7680e8dc2ec61481e","externalIds":{"MAG":"2766581718","DOI":"10.1016/j.freeradbiomed.2017.10.380","CorpusId":"3743468","PubMed":"29107745"},"title":"Respiratory syncytial virus induces NRF2 degradation through a promyelocytic leukemia protein ‐ ring finger protein 4 dependent pathway","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Inhibition of PML‐NB formation by blocking IFN pathway or silencing PML expression resulted in a significant reduction of RSV‐associated NRF2 degradation and increased antioxidant enzyme expression, identifying the RNF4‐PML pathway as a key regulator of antioxidant defenses in the course of viral infection."}},"tag":"EXP"},{"id":261,"details":{"paperId":"ce4e032a90e7d5728068fa611002a61fcc0ed93e","externalIds":{"MAG":"1981814313","DOI":"10.1111/cea.12498","CorpusId":"9458802","PubMed":"25627660"},"title":"Respiratory syncytial virus induces indoleamine 2,3‐dioxygenase activity: a potential novel role in the development of allergic disease","abstract":"Infants that develop severe bronchiolitis due to respiratory syncytial virus (RSV) are at increased risk of developing asthma later in life. We investigated a potential immunological mechanism for the association between RSV and the development of allergic inflammation. The enzyme indoleamine 2,3‐dioxygenase (IDO) has been reported to induce selective apoptosis of T helper 1 (Th1) cells and contributed to Th2‐biased immune responses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A potential immunological mechanism for the association between RSV and the development of allergic inflammation is investigated and the enzyme indoleamine 2,3‐dioxygenase (IDO) has been reported to induce selective apoptosis of T helper 1 (Th1) cells and contributed to Th2‐biased immune responses."}},"tag":"EXP"},{"id":407,"details":{"paperId":"ea53db23682188c475b07947c399e6084d1b90b8","externalIds":{"MAG":"2100278782","DOI":"10.1152/ajplung.90507.2008","CorpusId":"16711859","PubMed":"19525387"},"title":"Respiratory syncytial virus infection alters surfactant protein A expression in human pulmonary epithelial cells by reducing translation efficiency.","abstract":"Infection of neonatal lung by respiratory syncytial virus (RSV) is a common cause of respiratory dysfunction. Lung alveolar type II and bronchiolar epithelial (Clara) cells secrete surfactant protein A (SP-A), a collectin that is an important component of the pulmonary innate immune system. SP-A binds to the virus, targeting the infectious agent for clearance by host defense mechanisms. We have previously shown that while the steady-state level of SP-A mRNA increases approximately threefold after RSV infection, steady-state levels of cellular and secreted SP-A protein decrease 40-60% in human type II cells in primary culture, suggesting a mechanism where the virus alters components of the innate immune response in infected cells. In these studies, we find that changes in SP-A mRNA and protein levels in RSV-infected NCI-H441 cells (a bronchiolar epithelial cell line) recapitulate the results in SP-A expression observed in primary lung cells. While SP-A protein is normally ubiquitinated, there is no change in the level of SP-A protein ubiquitination or proteasome activity during RSV infection, suggesting that the reduced levels of SP-A protein are not due to degradation by activated proteasomes. SP-A mRNA is appropriately processed and exported from the nucleus to the cytoplasm during RSV infection. As evidenced by polysome analysis of SP-A mRNA and pulse-chase analysis of newly synthesized SP-A protein, we find a decrease in translational efficiency that is specific for SP-A mRNA. Therefore, the decrease in SP-A protein levels observed after RSV infection of pulmonary bronchiolar epithelial cells results from a mechanism that affects SP-A mRNA translation efficiency.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that changes in SP-A mRNA and protein levels in RSV-infected NCI-H441 cells (a bronchiolar epithelial cell line) recapitulate the results inSP-A expression observed in primary lung cells, suggesting a mechanism where the virus alters components of the innate immune response in infected cells."}},"tag":"EXP"},{"id":413,"details":{"paperId":"7a82a4322ca4a105ea0b5859ae518dffa13acd27","externalIds":{"MAG":"1581422023","DOI":"10.1159/000324777","CorpusId":"207673110","PubMed":"21865717"},"title":"Respiratory syncytial virus infection and the tight junctions of nasal epithelial cells.","abstract":"Respiratory syncytial virus (RSV) primarily infects upper respiratory tract cells, mainly nasal epithelial cells. The tight junctions of nasal epithelial cells are thought to perform important innate immune function against foreign materials including respiratory viruses. We investigated in vitro the relationship of RSV infection and the tight junctions of primary nasal epithelial cells which had been transfected with human telomerase reverse transcriptase (hTERT) to prolong cell life. Nasal epithelial cells developed tight junctions when cultured in medium containing fetal bovine serum, and these cells showed apparent resistance to RSV infection compared to control cells. RSV could infect these cells from apical but not basolateral side, suggesting that only apical side possess RSV receptor or a mechanism for absorbing RSV particles. Importantly, RSV infection of the cells enhanced the expression of tight junction proteins occludin, claudin-4 and ZO-1. These findings suggest that RSV infection induces polarity in the infected cells. This polarity could facilitate cellular secretion of propagated RSV, thereby spreading the infection.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In vitro RSV infection of nasal epithelial cells enhanced the expression of tight junction proteins occludin, claudin-4 and ZO-1, suggesting that polarity in the infected cells could facilitate cellular secretion of propagated RSV, thereby spreading the infection."}},"tag":"EXP"},{"id":63,"details":{"paperId":"95771231e6fe0485943cf67244c4ce5794dbf27f","externalIds":{"MAG":"1597991178","DOI":"10.1016/j.freeradbiomed.2015.05.043","CorpusId":"3979309","PubMed":"26073125"},"title":"Respiratory syncytial virus infection down-regulates antioxidant enzyme expression by triggering deacetylation-proteasomal degradation of Nrf2.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV-induced inhibition of Nrf2 activation, due to deacetylation and proteasomal degradation, could be targeted for therapeutic intervention aimed to increase airway antioxidant capacity during infection."}},"tag":"EXP"},{"id":44,"details":{"paperId":"1b63fba8b619f20d11322bd78ecc33e11d944720","externalIds":{"MAG":"1971841801","DOI":"10.1007/s705-002-8316-1","CorpusId":"11901751","PubMed":"11890521"},"title":"Respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is concluded that although RSV infection induces MMP-9, which can enhance the syncytia formation leading to RSV multiplication and spread it can be inhibited by MMP inhibitors."}},"tag":"EXP"},{"id":1,"details":{"paperId":"9509df42a454193c4616738e5ea6d8f254165e92","externalIds":{"MAG":"2151585934","DOI":"10.1002/cbin.10518","CorpusId":"40349549","PubMed":"26222045"},"title":"Respiratory syncytial virus infection inhibits TLR4 signaling via up‐regulation of miR‐26b","abstract":"Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illnesses in infants worldwide. TLR4 signal pathway plays a critical role in regulating immune response against RSV infection. However, the activation of TLR4 in RSV infection is still unclear. In present study, the expression levels of miR‐26b and TLR4 mRNA were detected in peripheral blood mononuclear cells (PBMCs) of children with or without RSV infected bronchiolitis. The expression levels of TLR4 and its downstream genes IFNβ and CCL5 were also quantified in PBMCs infected with RSVΔG or RSV A2 in vitro. The results showed that children with RSV infection had higher miR‐26b level and lower TLR4 mRNA level in PBMCs. miR‐26b was predicted to target TLR4. In vitro, miR‐26b mimic markedly down‐regulated TLR4 mRNA/protein expression and IFNβ/CCL5 concentrations while miR‐26b inhibitor up‐regulated these levels. This study reveals that RSV infection inhibits TLR4 signaling via up‐regulation of miR‐26b, which provides a potential therapeutic target for preventing and treating RSV infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"RSV infection inhibits TLR4 signaling via up‐regulation of miR‐26b, which provides a potential therapeutic target for preventing and treating RSV infection."}},"tag":"EXP"},{"id":505,"details":{"paperId":"eb71ec0a6abc8880fa6646e81cacee17a0541306","externalIds":{"MAG":"2083390198","DOI":"10.3892/mmr.2014.2357","CorpusId":"6535148","PubMed":"24968899"},"title":"Respiratory syncytial virus infection modulates interleukin‑8 production in respiratory epithelial cells through a transcription factor‑activator protein‑1 signaling pathway.","abstract":"Respiratory syncytial virus (RSV) is a leading cause of respiratory duct infection that can result in severe clinical symptoms, particularly among children under 3 years of age. In the current study, the effect of RSV on airway epithelial cell function and the potential signaling pathways involved were investigated. A549 human airway epithelial cells were infected with RSV at a multiplicity of infection of 1. After 24 h, interleukin (IL)‑8 secretion in the cell supernatant was analyzed. A microarray assay of RSV‑infected A549 cells was conducted in order to identify any potential pathways involved, and quantitative polymerase chain reaction was performed to examine mRNA expression levels in these pathways. Electrophoretic mobility shift assays of nuclear transcription factors were conducted for further verification. IL‑8 levels increased significantly in the supernatant of RSV‑infected A549 cells compared with levels in non‑infected cells. Microarray data suggested the involvement of the Toll‑like receptor 4 (TLR4) pathway, and mRNA expression levels of genes (MYD88, TRAM and TRIF) involved in this pathway were higher in infected cells. Enhanced synthesis of activator protein‑1 (AP‑1) was observed. RSV infection of A549 cells may promote IL‑8 secretion. In conclusion, the results of the present study indicate that the TLR4 signaling pathway, in conjunction with MYD88, TRAM, TRIF and the transcription factor AP‑1, may activate immune responses to RSV infection in airway epithelial cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results of the present study indicate that the TLR4 signaling pathway, in conjunction with MYD88, TRAM, TRIF and the transcription factor AP‑1, may activate immune responses to RSV infection in airway epithelial cells."}},"tag":"EXP"},{"id":37,"details":{"paperId":"37399b6bf8861a97805bb95940a4b3a3b7f51b72","externalIds":{"DOI":"10.1007/s00705-018-3739-4","CorpusId":"254054370"},"title":"Respiratory syncytial virus infection up-regulates TLR7 expression by inducing oxidative stress via the Nrf2/ARE pathway in A549 cells","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The results indicate that TLR7 up-regulation is related to RSV infection and the induction of oxidative stress and thatTLR7 expression was mediated by the anti-inflammatory effects of Nrf2/ARE pathway inhibitors or agonists."}},"tag":"EXP"},{"id":329,"details":{"paperId":"1c100b7c1bf3ed5c3d97892020cc93ff21b0046c","externalIds":{"MAG":"2115284965","DOI":"10.1128/jvi.46.2.667-672.1983","CorpusId":"19189344","PubMed":"6682456"},"title":"Respiratory syncytial virus mRNA coding assignments","abstract":"The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively."}},"tag":"EXP"},{"id":253,"details":{"paperId":"15db15deec9e5a343306071bb97b1195fb2313f7","externalIds":{"DOI":"10.1101/2021.10.13.464285","CorpusId":"239021215"},"title":"Respiratory syncytial virus matrix protein assembles as a lattice with local and extended order that coordinates the position of the fusion glycoprotein","abstract":"Respiratory syncytial virus (RSV) is a significant cause of respiratory illness in young children and adults worldwide. There is currently no vaccine or targeted antiviral for RSV. RSV is an enveloped, filamentous, negative-strand RNA virus. Individual virions vary in both diameter and length, with an average diameter of ∼130 nm and ranging from ∼500 nm to over 10 μm in length. The RSV matrix (M) protein is peripherally associated with the interior of the viral membrane. Though the general arrangement of structural proteins within the virion is known, the molecular organization of M and other structural proteins was previously unknown. Here, using whole-cell cryo-electron tomography and sub-tomogram averaging, we show that M is arranged in a packed helical-like lattice of M-dimers ordered at an angle of ∼47° to the viral long axis. Sub-tomogram averages including F and M indicate that the position of F on the viral surface is correlated with the underlying M lattice. Finally, we report that RSV F is frequently observed as pairs, with the F trimers oriented in an anti-parallel conformation to support potential interaction between trimers. These results provide insight into RSV assembly and virion organization and may aid in the identification and development of RSV vaccines and anti-viral targets.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Insight is provided into RSV assembly and virion organization and this work reports that RSV F is frequently observed as pairs, with the F trimers oriented in an anti-parallel conformation to support potential interaction between trimers."}},"tag":"EXP"},{"id":230,"details":{"paperId":"c4088a9b2aa339846f99d1645331f8e22e02bf7d","externalIds":{"MAG":"2143338842","DOI":"10.1099/0022-1317-83-4-753","CorpusId":"17162993","PubMed":"11907323"},"title":"Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells.","abstract":"Little is known about the functions of the matrix (M) protein of respiratory syncytial virus (RSV). By analogy with other negative-strand RNA viruses, the M protein should inhibit the viral polymerase prior to packaging and facilitate virion assembly. In this study, localization of the RSV M protein in infected cells and its association with the RSV nucleocapsid complex was investigated. RSV-infected cells were shown to contain characteristic cytoplasmic inclusions. Further analysis showed that these inclusions were localization sites of the M protein as well as the N, P, L and M2-1 proteins described previously. The M protein co-purified with viral ribonucleoproteins (RNPs) from RSV-infected cells. The transcriptase activity of purified RNPs was enhanced by treatment with antibodies to the M protein in a dose-dependent manner. These data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription."}},"tag":"EXP"},{"id":341,"details":{"paperId":"51f02858e46b2b5438b7099b92a52bd2139eed2d","externalIds":{"MAG":"2128049245","DOI":"10.1128/jvi.69.4.2667-2673.1995","CorpusId":"9629703","PubMed":"7884920"},"title":"Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells","abstract":"Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that RS virus matures at the apical surface of polarized epithelial cell lines, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus."}},"tag":"EXP"},{"id":56,"details":{"paperId":"09b5e705edb46e535b2482ce3072ee971c303a84","externalIds":{"MAG":"2513797372","DOI":"10.1016/j.bbrc.2016.08.142","CorpusId":"22935970","PubMed":"27569280"},"title":"Respiratory syncytial virus non-structural protein 1 facilitates virus replication through miR-29a-mediated inhibition of interferon-α receptor.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results suggest that miR-29a, upregulated during RSV infection, is a negative regulator of IFNAR1 and is critical for RSV NS1-induced virus replication."}},"tag":"EXP"},{"id":71,"details":{"paperId":"83b27b4fc3a3e30931a37d8418c46872f5e39b2b","externalIds":{"DOI":"10.1016/j.molimm.2021.07.019","CorpusId":"236637540","PubMed":"34332182"},"title":"Respiratory syncytial virus nonstructural protein 1 breaks immune tolerance in mice by downregulating Tregs through TSLP-OX40/OX40L-mTOR axis.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data indicate that RSV NS1 protein breaks immune tolerance and induces airway inflammation and hyperresponsiveness in mice and is at least partially mediated by modulating AKT-mTOR signaling pathways."}},"tag":"EXP"},{"id":83,"details":{"paperId":"4ed90180d7a1ae4cd609bd0b36b9bb0928afaa65","externalIds":{"MAG":"2023594156","DOI":"10.1016/J.VIROL.2005.09.009","CorpusId":"29135263","PubMed":"16216295"},"title":"Respiratory syncytial virus nonstructural protein 2 specifically inhibits type I interferon signal transduction.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results indicate that NS2 regulates Stat2 levels during RSV infection, thereby modulating viral effects on interferon-dependent gene expression."}},"tag":"EXP"},{"id":458,"details":{"paperId":"eb2ffe20bfc608e26f0c6e7700f0e70f410baf92","externalIds":{"PubMedCentral":"6797084","MAG":"2981054348","DOI":"10.1371/journal.ppat.1007984","CorpusId":"204774082","PubMed":"31622448"},"title":"Respiratory syncytial virus nonstructural proteins 1 and 2: Exceptional disrupters of innate immune responses","abstract":"Human respiratory syncytial virus (RSV) is the most important cause of acute lower respiratory tract disease in infants worldwide. As a first line of defense against respiratory infections, innate immune responses, including the production of type I and III interferons (IFNs), play an important role. Upon infection with RSV, multiple pattern recognition receptors (PRRs) can recognize RSV-derived pathogen-associated molecular patterns (PAMPs) and mount innate immune responses. Retinoic-acid-inducible gene-I (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) have been identified as important innate receptors to mount type I IFNs during RSV infection. However, type I IFN levels remain surprisingly low during RSV infection despite strong viral replication. The poor induction of type I IFNs can be attributed to the cooperative activity of 2 unique, nonstructural (NS) proteins of RSV, i.e., NS1 and NS2. These viral proteins have been shown to suppress both the production and signaling of type I and III IFNs by counteracting a plethora of key host innate signaling proteins. Moreover, increasing numbers of IFN-stimulated genes (ISGs) are being identified as targets of the NS proteins in recent years, highlighting an underexplored protein family in the identification of NS target proteins. To understand the diverse effector functions of NS1 and NS2, Goswami and colleagues proposed the hypothesis of the NS degradasome (NSD) complex, a multiprotein complex made up of, at least, NS1 and NS2. Furthermore, the crystal structure of NS1 was resolved recently and, remarkably, identified NS1 as a structural paralogue of the RSV matrix protein. Unfortunately, no structural data on NS2 have been published so far. In this review, we briefly describe the PRRs that mount innate immune responses upon RSV infection and provide an overview of the various effector functions of NS1 and NS2. Furthermore, we discuss the ubiquitination effector functions of NS1 and NS2, which are in line with the hypothesis that the NSD shares features with the canonical 26S proteasome.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The PRRs that mount innate immune responses upon RSV infection are described and the ubiquitination effector functions of NS1 and NS2 are discussed, in line with the hypothesis that the NSD shares features with the canonical 26S proteasome."}},"tag":"EXP"},{"id":51,"details":{"paperId":"c294c55961cf8dac462abaa9f45077171234a098","externalIds":{"MAG":"1996022663","DOI":"10.1016/0168-1702(94)90003-5","CorpusId":"4161397","PubMed":"7513931"},"title":"Respiratory syncytial virus nucleocapsid protein (N) expressed in insect cells forms nucleocapsid-like structures.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Nucleocapsid structures similar to those purified from RSV-infected cells could be observed by electron microscopy after negative staining of cellular extracts."}},"tag":"EXP"},{"id":206,"details":{"paperId":"8a77c9a9a51a566cc719c2535c0d1d67cd579899","externalIds":{"MAG":"1967788376","DOI":"10.1099/0022-1317-66-3-417","CorpusId":"23765367","PubMed":"3919149"},"title":"Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins.","abstract":"The cell-associated glycoproteins of respiratory syncytial (RS) virus included GP1 (90K), VP70 (70K), VGP48 (48K) and GP26 (26K). Although present in infected cells, there was no VP70 in purified virus. Trypsin treatment of infected cells removed 80 to 90% of VP70 as well as its products VGP48 and GP26. This suggested that most of the VP70 in the cell is located on the plasma membrane. The glycoproteins of purified RS virus (GP1, VGP48 and GP26) contain mannose, galactose and fucose as well as glucosamine, but the quantity of mannose in GP1 is low when compared to that of the other three sugars. The effects that follow the treatment of infected cells with the glycosylation inhibitors tunicamycin and monensin, and the treatment of the immunoprecipitated product of pulse-chase experiments with endonuclease H demonstrated that VP70 and its products contained N-linked oligosaccharides, and that the oligosaccharides of the mature VGP48 subunit were of the complex type, while GP1 contained both N- and O-linked oligosaccharides. The non-glycosylated forms of VP70 and GP1 have estimated mol. wt. of 50K and 33K respectively. Therefore, the carbohydrate contribution to the mol. wt. of VP70 and GP1, as determined by PAGE, was equivalent to 20K for the former and 57K for the latter. The majority of the GP1 oligosaccharides were O-linked, a form of sugar linkage not previously found among paramyxoviruses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The effects that follow the treatment of infected cells with the glycosylation inhibitors tunicamycin and monensin, and thetreatment of the immunoprecipitated product of pulse-chase experiments with endonuclease H demonstrated that VP70 and its products contained N-linked oligosaccharides, and that the oligosACcharides of the mature VGP48 subunit were of the complex type."}},"tag":"EXP"},{"id":425,"details":{"paperId":"5cb67b8103fed813182562e341dd55edb81c4998","externalIds":{"DOI":"10.1186/s12929-018-0416-6","CorpusId":"255863828"},"title":"Respiratory syncytial virus prolifically infects N2a neuronal cells, leading to TLR4 and nucleolin protein modulations and RSV F protein co-localization with TLR4 and nucleolin","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, Fprotein expression, RSV viral titers, and late apoptotic levels of N 2a cells."}},"tag":"EXP"},{"id":234,"details":{"paperId":"4fa2a76710271a60150e7888355a76cfac66595b","externalIds":{"MAG":"2922435764","DOI":"10.1099/jmm.0.000959","CorpusId":"73464359","PubMed":"30843783"},"title":"Respiratory syncytial virus promoted the differentiation of Th17 cells in airway microenvironment through activation of Notch-1/Delta3.","abstract":"BACKGROUND\nRespiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. Infection of RSV has been shown to induce Th lymphocyte differentiation. The present study was designed to determine the effects of RSV on the expression of Notch-1 and the related mechanisms on subsequent differentiation of Th lymphocytes.\n\n\nMETHODS\nA RSV-infected animal model was established and investigated at 7, 28 and 60 days post infection. Real-time qPCR and Western blot were used to observe the expression levels of Notch-1 in CD4+ T cells and its five ligands in lung tissues. The methylation levels of CpG islands in autoimmune regulator (AIRE) and Notch-1 promoters were analysed by time-of-flight mass spectrometry. The differentiation of Th lymphocytes was assayed by real-time qPCR. The distribution of JAG1 and DLL3 in the lung tissues were assayed by immunohistochemistry. The correlation between Th17 and DLL3 was analysed by simple correlation.\n\n\nRESULTS\nThe results showed that RSV promoted the expression and de-methylation of Notch-1 promoters in CD4+ T cells. Moreover, RSV infection promoted Th1 differentiation at day 7 and day 28; Th17 differentiation at day 7, day 28 and day 60; Th2 differentiation at day 28 and day 60. At the same time, RSV infection promoted the expression of JAG1 and DLL3. Activation of Notch-1/ DLL 3 in lungs may be associated with the differentiation of Th17 lymphocytes.\n\n\nCONCLUSIONS\nOur data showed that activation of RSV stimulated the differentiation of Th17 in airway microenvironment through activation Notch-1/DLL3, which may be associated with the occurrence and development of RSV-induced asthma.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data showed that activation of RSV stimulated the differentiation of Th17 in airway microenvironment through activation Notch-1/DLL3, which may be associated with the occurrence and development ofRSV-induced asthma."}},"tag":"EXP"},{"id":193,"details":{"paperId":"aaab3531b2b89ce18035d720bf4cdd94795f6334","externalIds":{"MAG":"1990167513","DOI":"10.1089/vim.2008.0098","CorpusId":"13134974","PubMed":"19435411"},"title":"Respiratory syncytial virus proteins modulate suppressors of cytokine signaling 1 and 3 and the type I interferon response to infection by a toll-like receptor pathway.","abstract":"Respiratory syncytial virus (RSV) is a common cause of repeat infections throughout life and potentially severe lower respiratory tract illness in infants, young children, and the elderly. RSV proteins have been shown to contribute to immune evasion by several means, including modification of cytokine and chemokine responses whose expression is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. In this study, we examine the role of SOCS1 and SOCS3 regulation of the type I interferon (IFN) response in normal fully-differentiated human bronchial epithelial cells infected with RSV or with an RSV mutant virus lacking the G gene. The results show that RSV G protein modulates SOCS expression to inhibit type I IFN and interferon-stimulated gene (ISG)-15 expression very early as well as late in infection, and that SOCS induction is linked to toll-like receptor (TLR) signaling by RSV F protein, as indicated by interferon-regulatory factor (IRF)-3 activation and nuclear translocation. These findings indicate that RSV surface proteins signal through the TLR pathway, suggesting that this may be an important mechanism to reduce type I IFN expression to aid virus replication.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is indicated that RSV surface proteins signal through the TLR pathway, suggesting that this may be an important mechanism to reduce type I IFN expression to aid virus replication."}},"tag":"EXP"},{"id":466,"details":{"paperId":"cabe0a2ff1420a833717a9a5c11f82470435330b","externalIds":{"PubMedCentral":"9262236","DOI":"10.1371/journal.ppat.1010619","CorpusId":"250336339","PubMed":"35797399"},"title":"Respiratory syncytial virus ribonucleoproteins hijack microtubule Rab11 dependent transport for intracellular trafficking","abstract":"Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway."}},"tag":"EXP"},{"id":155,"details":{"paperId":"1c5c579c5fffcff38621c8235264bee7156c056d","externalIds":{"MAG":"2081789829","DOI":"10.1073/pnas.0712144105","CorpusId":"20474689","PubMed":"18621683"},"title":"Respiratory syncytial virus uses a Vps4-independent budding mechanism controlled by Rab11-FIP2","abstract":"Respiratory syncytial virus (RSV) infects polarized epithelia, which have tightly regulated trafficking because of the separation and maintenance of the apical and basolateral membranes. Previously we established a link between the apical recycling endosome (ARE) and the assembly of RSV. The current studies tested the role of a major ARE-associated protein, Rab11 family interacting protein 2 (FIP2) in the virus life cycle. A dominant-negative form of FIP2 lacking its N-terminal C2 domain reduced the supernatant-associated RSV titer 1,000-fold and also caused the cell-associated virus titer to increase. These data suggested that the FIP2 C2 mutant caused a failure at the final budding step in the virus life cycle. Additionally, truncation of the Rab-binding domain from FIP2 caused its accumulation into mature filamentous virions. RSV budding was independent of the ESCRT machinery, the only well-defined budding mechanism for enveloped RNA viruses. Therefore, RSV uses a virus budding mechanism that is controlled by FIP2.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Testing the role of a major ARE-associated protein, Rab11 family interacting protein 2 (FIP2) in the virus life cycle suggested that the FIP2 C2 mutant caused a failure at the final budding step in theirus life cycle."}},"tag":"EXP"},{"id":186,"details":{"paperId":"2ba968dea9349f112c75df965540d92886389ccb","externalIds":{"MAG":"2900694504","DOI":"10.1080/14712598.2018.1544239","CorpusId":"53304125","PubMed":"30426788"},"title":"Respiratory syncytial virus vaccine: where are we now and what comes next?","abstract":"ABSTRACT Introduction: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants and elderly and to date, there is no safe or effective vaccine against RSV. Areas Covered: This review provides a roadmap to RSV vaccine development. It is a journey spanning over more than half a century from the initial disappointment with inactivated formalin vaccine to the current advancements in vaccine technology. We highlight the important aspects of RSV structural biology and protective immune response. We include discussion of newer fusion glycoprotein immune targets and current vaccine candidates. We used Pub Med and Medline resources for literature search. Expert opinion: A resurgence of information on the burden related to RSV infection coupled with the newer understanding of the molecular mechanism of RSV infection has reignited a tremendous activity in RSV vaccine discovery. The vaccine pipeline is diverse and target populations are varied, thus making the goal of a safe and effective RSV vaccine in the future within reach.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This review provides a roadmap to RSV vaccine development, highlighting the important aspects of RSV structural biology and protective immune response and including discussion of newer fusion glycoprotein immune targets and current vaccine candidates."}},"tag":"DRUG"},{"id":21,"details":{"paperId":"977e839f17da698c56d7990143e8b22672eb4130","externalIds":{"MAG":"2081356514","DOI":"10.1006/VIRO.2001.1340","CorpusId":"46015699","PubMed":"12009871"},"title":"Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus.","abstract":"Cell surface glycosaminoglycans (GAGs) are responsible for the majority of respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the viral attachment protein, but more recently virus lacking the G protein was shown to be infectious in cell culture. We have compared the GAG dependence of a recombinant, green fluorescent protein-expressing virus containing the F protein as its only viral glycoprotein (rgRSV-F) to isogenic complete virus containing all three viral glycoproteins (rgRSV-SGF). Attachment and infection by each virus was found to be largely dependent on cell surface heparan sulfate (HS) based on the finding that both activities were inhibited by preincubation of virus with soluble HS, by removal of HS from target cells by enzymatic treatment or mutation, or by pretreatment of the target cells with basic fibroblast growth factor (bFGF), which binds HS. These results, coupled with the previous finding that SH is not involved in virion binding (S. Techaarpornkul, N. Barretto, and M. Peeples, 2001, J. Virol. 75, 6825-6834), suggest that, in the context of the virion, both the G and F proteins bind to HS. Interestingly, both rgRSV-F and rgRSV-SGF retained significant binding activity and infectivity despite these treatments, suggesting an alternate productive attachment and infection pathway. This property of GAG independence was particularly apparent for rgRSV-F virions, which retained nearly half of its attachment and infection activities in most of these experiments. Comparison of the attachment and infection activities of rgRSV-SGF and rgRSV-F virions with a Chinese hamster ovary cell line and a derivative thereof that is defective in GAG synthesis indicated that approximately 50% of rgRSV-SGF attachment is due to G protein-GAG binding, 25% to F protein-GAG binding, and 25% to an independent pathway. This alternative pathway presumably is mediated by the sole remaining viral surface protein, F, although the formal possibility exists that some other virion-associated protein is involved.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Compared with a recombinant, green fluorescent protein-expressing virus containing the F protein as its only viral glycoprotein, both the G and F proteins bind to HS, suggesting an alternate productive attachment and infection pathway in the context of the virion."}},"tag":"EXP"},{"id":151,"details":{"paperId":"caad0169ab3f9230183c75f36e81e42b02de95eb","externalIds":{"MAG":"1997527149","DOI":"10.1053/JINF.2002.1015","CorpusId":"21339621","PubMed":"12217726"},"title":"Respiratory syncytial virus--viral biology and the host response.","abstract":"Respiratory syncytial virus (RSV) is the most important cause of respiratory tract infection in infants. We have an incomplete understanding of the reasons why some infants are more severely affected by RSV than others. There is no effective antiviral treatment for the infection. Advances in our understanding of the biology of RSV, particularly in relation to the attachment protein G and the fusion protein F, have revealed potential targets for new antiviral therapies and vaccine development. In response to RSV infection an intense inflammatory response is triggered, mediated initially by the infected airway epithelial cells. Cell mediated responses are important in controlling the extent of infection and in viral clearance. Humoral responses are important in protection. There is early evidence that genetic variation of the host response can influence the outcome of RSV-induced bronchiolitis.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Advances in the understanding of the biology of RSV, particularly in relation to the attachment protein G and the fusion protein F, have revealed potential targets for new antiviral therapies and vaccine development."}},"tag":"EXP"},{"id":211,"details":{"paperId":"a85be7d1eff5b19b9c9637175671659a9f8743b4","externalIds":{"MAG":"2001256669","DOI":"10.1099/0022-1317-68-6-1749","CorpusId":"36828713","PubMed":"3035067"},"title":"Respiratory virus infection of peripheral blood monocytes: correlation with ageing of cells and interferon production in vitro.","abstract":"The ability of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) to replicate in peripheral blood monocytes cultured in vitro for 1, 2, 4 or 7 days prior to infection was investigated. Inoculation of 1-day old monocytes produced at least tenfold less new virus than infection of the older, more macrophage-like cells for both viruses. PIV3 induced extensive syncytium formation, whereas RSV caused a cytopathic effect manifest by increased rounding of the cells with minimal syncytium formation. Supernatants of infected monocytes were assayed for human interferon-alpha (HuIFN-alpha) in an attempt to explain the restricted viral replication in the youngest monocytes. In PIV3-infected cells, HuIFN-alpha production was inversely correlated with new virus formation. Monocytes infected after 1 day in culture produced 800 IU/ml of HuIFN-alpha; the older cells produced 100 to 200 IU/ml. In contrast, monocytes infected on day 1 with RSV produced minimal amounts (1.5 IU/ml) of HuIFN-alpha. Increasing amounts of HuIFN-alpha were detected in cells infected with RSV after 2, 4 or 7 days in culture, reaching a maximum of 400 IU/ml on day 7. Further investigation of the apparent restriction of replication in young monocyte cultures may be helpful in understanding the pathogenesis of these respiratory infections.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The ability of respiratory syncytial virus and parainfluenza virus type 3 to replicate in peripheral blood monocytes cultured in vitro for 1, 2, 4 or 7 days prior to infection was investigated."}},"tag":"EXP"},{"id":196,"details":{"paperId":"9b0c98d4ed0e4f3957ebae49bb5ffccc20c48f4c","externalIds":{"PubMedCentral":"9749005","DOI":"10.1093/infdis/jiac295","CorpusId":"250731151","PubMed":"35861054"},"title":"Retinoic Acid–Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection","abstract":"Abstract Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid–inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an “antiviral state.” In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication, and a single intravenous injection of 3pRNA 1 day beforeRSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues."}},"tag":"EXP"},{"id":361,"details":{"paperId":"1274a525ec9b32f06da312e9b60a16f45b7930c5","externalIds":{"MAG":"1886359527","DOI":"10.1128/JVI.73.9.7262-7270.1999","CorpusId":"44654020","PubMed":"10438814"},"title":"RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation","abstract":"ABSTRACT The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that Rho a can facilitate RSV-induced syncyTium formation."}},"tag":"EXP"},{"id":394,"details":{"paperId":"85294b79c1b477a7a90c873cd109e92c20f3d6d6","externalIds":{"MAG":"2140228548","DOI":"10.1128/JVI.79.9.5326-5336.2005","CorpusId":"43086110","PubMed":"15827147"},"title":"RhoA Signaling Is Required for Respiratory Syncytial Virus-Induced Syncytium Formation and Filamentous Virion Morphology","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion."}},"tag":"EXP"},{"id":494,"details":{"paperId":"6966d04a46f765deece1a0136e9d7c9b6c972915","externalIds":{"PubMedCentral":"8781601","DOI":"10.3390/pathogens11010026","CorpusId":"245532480","PubMed":"35055974"},"title":"Role of ARP2/3 Complex-Driven Actin Polymerization in RSV Infection","abstract":"Respiratory syncytial virus (RSV) is the leading viral agent causing bronchiolitis and pneumonia in children under five years old worldwide. The RSV infection cycle starts with macropinocytosis-based entry into the host airway epithelial cell membrane, followed by virus transcription, replication, assembly, budding, and spread. It is not surprising that the host actin cytoskeleton contributes to different stages of the RSV replication cycle. RSV modulates actin-related protein 2/3 (ARP2/3) complex-driven actin polymerization for a robust filopodia induction on the infected lung epithelial A549 cells, which contributes to the virus’s budding, and cell-to-cell spread. Thus, a comprehensive understanding of RSV-induced cytoskeletal modulation and its role in lung pathobiology may identify novel intervention strategies. This review will focus on the role of the ARP2/3 complex in RSV’s pathogenesis and possible therapeutic targets to the ARP2/3 complex for RSV.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A comprehensive understanding of RSV-induced cytoskeletal modulation and its role in lung pathobiology may identify novel intervention strategies and possible therapeutic targets to the ARP2/3 complex for RSV."}},"tag":"EXP"},{"id":382,"details":{"paperId":"c630b42b55683b46163426d88d614dcc02fc1e69","externalIds":{"MAG":"2152604492","DOI":"10.1128/JVI.77.3.1747-1756.2003","CorpusId":"6527160","PubMed":"12525608"},"title":"Role of Plasma Membrane Lipid Microdomains in Respiratory Syncytial Virus Filament Formation","abstract":"ABSTRACT The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"An overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation."}},"tag":"EXP"},{"id":16,"details":{"paperId":"8926d826189a74b4bae6a3c1c862b45c699d111c","externalIds":{"MAG":"1974333324","DOI":"10.1006/VIRO.1998.9471","CorpusId":"37955229","PubMed":"9875324"},"title":"Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus.","abstract":"Cytoskeletal protein actin and nonactin cellular proteins were essential for human respiratory syncytial virus (RSV) gene expression. In vitro, specific antibodies against actin inhibited RSV transcription, whereas antibodies against other cytoskeletal proteins had little or no effect. Affinity purified cellular actin or bacterially expressed recombinant actin activated RSV transcription. However, optimal transcription required additional cellular protein(s) that appeared to function as accessory factor(s) for actin. In the absence of actin, these proteins did not activate viral transcription. Purified viral nucleocapsids contained actin, but no cytokeratin, tubulin, or vimentin. Cytochalasin D or DNasel--agents that destabilize actin polymers--had little effect on RSV transcription. RSV infection itself seemed to alter the structure of the cellular actin filaments. Treatment of infected cells with cytochalasin D produced a more severe disruption of the filaments and drastically reduced the production of infectious virus particles but still had little effect on intracellular synthesis of viral macromolecules. Thus actin seems to serve a dual role in RSV life cycle: its monomeric form as well as polymeric form activate viral transcription, while only the microfilament form may take part in viral morphogenesis and/or budding.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Actin seems to serve a dual role in RSV life cycle: its monomeric form as well as polymeric form activate viral transcription, while only the microfilament form may take part in viral morphogenesis and/or budding."}},"tag":"EXP"},{"id":46,"details":{"paperId":"700af18cff70d0cb313da2c091d454ab04ed766b","externalIds":{"MAG":"2052402839","PubMedCentral":"7130872","DOI":"10.1016/0042-6822(88)90560-0","CorpusId":"25546234","PubMed":"3369089"},"title":"Role of oligosaccharides in the structure and function of respiratory syncytial virus glycoproteins","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Virus infectivity was sensitive to limited removal of N-linked or O-linked oligosaccharides by endoglycosidases under conditions which did not greatly alter the molecular weight of the G protein."}},"tag":"EXP"},{"id":358,"details":{"paperId":"d3c49a269b8621920cb17cd28034d960da32f1dd","externalIds":{"MAG":"2104236554","DOI":"10.1128/JVI.73.7.5852-5864.1999","CorpusId":"22208314","PubMed":"10364337"},"title":"Role of the M2-1 Transcription Antitermination Protein of Respiratory Syncytial Virus in Sequential Transcription","abstract":"ABSTRACT M2-1 protein of human respiratory syncytial virus (RSV) is a transcription antitermination factor that is important for the efficient synthesis of full-length mRNAs as well as for the synthesis of polycistronic readthrough mRNAs, which are characteristic of nonsegmented negative-strand RNA viruses. The contributions of these effects to RSV sequential transcription were investigated with minigenomes which contained one to five genes which were either foreign marker genes or authentic RSV genes. When evaluated on a promoter-proximal gene, the effect of M2-1 on the synthesis of full-length mRNA was much greater for a long (1,212- or 1,780-nucleotide) gene (up to a 615-fold increase) than for a short (274-nucleotide) gene (less than a 2-fold increase). This was independent of whether the gene contained non-RSV or RSV-specific sequence. Once the polymerase had terminated prematurely, it was unable to reinitiate at a downstream gene. These studies also confirmed that M2-1 enhances the synthesis of polycistronic mRNAs and that the magnitude of this effect varied greatly among different naturally occurring gene junctions. The synthesis of polycistronic mRNAs, which presumably involves antitermination at the gene-end signal, required a higher level of M2-1 than did the synthesis of the corresponding monocistronic mRNAs. M2-1 did not have a comparable antitermination effect at the junction between the leader region and the first gene. In a minigenome containing the NS1 and NS2 genes in their authentic sequence context, synthesis of full-length NS1 and NS2 mRNAs in the absence of M2-1 was remarkably high (36 and 57%, respectively, of the maximum levels observed in the presence of M2-1). In contrast, synthesis of mRNA from additional downstream genes was highly dependent on M2-1. Thus, RSV has the potential for two transcription programs: one in the absence of M2-1, in which only the NS1 and NS2 genes are transcribed, and one in the presence of M2-1, in which sequential transcription of the complete genome occurs. The dependence on M2-1 for transcription was greater for a gene in the fifth position from the promoter than for one in the third position. This indicates that under conditions where M2-1 is limiting, its concentration affects the gradient of transcription. Although M2-1 was found to have profound effects on transcription, it had no effect on replication of any minigenome tested, suggesting that it is not an active participant in RNA replication or regulation of RNA replication. Finally, since a permissive RSV infection is marked by a gradual increase in the intracellular accumulation of viral proteins including M2-1, we examined the relative abundances of various mRNAs during RSV infection for evidence of temporal regulation of transcription. None was found, implying that the availability of M2-1 during a permissive infection is sufficient at all times such that its concentration does not mediate temporal regulation of gene transcription.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Although M2-1 was found to have profound effects on transcription, it had no effect on replication of any minigenome tested, suggesting that it is not an active participant in RNA replication or regulation of RNA replication."}},"tag":"EXP"},{"id":475,"details":{"paperId":"3088aaedef9164b562bbe45f522ddbb6e532e42a","externalIds":{"MAG":"3147748326","DOI":"10.26502/ACBR.50170159","CorpusId":"234161042"},"title":"Roles of Human Respiratory Syncytial Virus Proteins in Immune Responses","abstract":"Human respiratory syncytial virus (hRSV) has evaded active vaccination or effective antiviral therapy for decades and continues to be the leading cause of morbidity and hospitalization in infants, the elderly, and the immunocompromised worldwide. Inadequate understanding of the antigenic intricacies of its viral proteins and the immune responses they generate in the host is the greatest obstacle to the progress of hRSV prevention and treatment. Currently, the prefusion F protein is considered the most effective antigen for inducing protective immunity. Other molecular components of hRSV, such as the G or N proteins, have also been explored as potential targets for disease control. However, important knowledge gaps remain about the role played by various hRSV proteins in immunobiology and pathology. This review summarizes the unique immunomodulatory aspects of hRSV infection, the viral proteins involved in intracellular immune signaling, and the viral interactions in play with the host’s immune system. Arch Clin Biomed Res 2021; 5 (2): 185-200 DOI: 10.26502/acbr.50170159 Archives of Clinical and Biomedical Research Vol. 5 No.2 – April 2021. [ISSN 2572-9292]. 186","publicationTypes":["Review"],"tldr":{"model":"tldr@v2.0.0","text":"The unique immunomodulatory aspects of hRSV infection, the viral proteins involved in intracellular immune signaling, and the viral interactions in play with the host’s immune system are summarized."}},"tag":"EXP"},{"id":247,"details":{"paperId":"49c7b0adbfc8d97b78d1d3849bc17c9640ad80a4","externalIds":{"MAG":"2126523170","DOI":"10.1099/VIR.0.81715-0","CorpusId":"2307537","PubMed":"16690930"},"title":"Sequence elements of the fusion peptide of human respiratory syncytial virus fusion protein required for activity.","abstract":"We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein (F(TM-)) representing the ectodomain of the full-length F. F(TM-) molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the F(TM-) trimer leads to a change in shape from cones to lollipops that aggregate into rosettes. This aggregation apparently occurs by interaction of the fusion peptides of F(TM-) molecules that are exposed after cleavage. Since exposure of the fusion peptide is a key event in the process of membrane fusion, changes associated with F(TM-) cleavage may reflect those occurring in full-length F during membrane fusion. Deletions or substitutions that changed either the length, charge or hydrophobicity of the fusion peptide inhibited aggregation of F(TM-), and these mutants remained as unaggregated cones after cleavage. In contrast, more conservative changes did not inhibit the change of shape and aggregation of F(TM-). When the same changes were introduced in the fusion peptide of full-length F, only the mutations that inhibited aggregation of F(TM-) prevented membrane fusion. Thus, the conformational changes that follow completion of cleavage of the F(TM-) protein require a functional fusion peptide. These sequence constraints may restrict accumulation of sequence changes in the fusion peptide of HRSV F when compared with other hydrophobic regions of the molecule.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The conformational changes that follow completion of cleavage of the F(TM-) protein require a functional fusion peptide, and sequence constraints may restrict accumulation of sequence changes in the fusion peptides of HRSV F when compared with other hydrophobic regions of the molecule."}},"tag":"EXP"},{"id":320,"details":{"paperId":"1e523fb81e29e09c2a056e791b10bf057110041c","externalIds":{"MAG":"2154640353","DOI":"10.1128/JVI.02780-06","CorpusId":"46015904","PubMed":"17494077"},"title":"Small Interfering RNA Profiling Reveals Key Role of Clathrin-Mediated Endocytosis and Early Endosome Formation for Infection by Respiratory Syncytial Virus","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections in infants and the elderly. Like many other pH-independent enveloped viruses, RSV is thought to enter at the cell surface, independently of common endocytic pathways. We have used a targeted small interfering RNA (siRNA) library to identify key cellular genes involved in cytoskeletal dynamics and endosome trafficking that are important for RSV infection. Surprisingly, RSV infection was potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis, including clathrin light chain. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. We conclude that, while RSV may be competent to enter at the cell surface, clathrin function and endocytosis are a necessary and important part of a productive RSV infection, even though infection is strictly independent of pH. These findings raise the possibility that other pH-independent viruses may share a similar dependence on endocytosis for infection and provide a new potential avenue for treatment of infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is concluded that, while RSV may be competent to enter at the cell surface, clathrin function and endocytosis are a necessary and important part of a productive RSV infection, even though infection is strictly independent of pH."}},"tag":"EXP"},{"id":200,"details":{"paperId":"e22dea6622f0126b768cbe3a921ae54bca2c0096","externalIds":{"MAG":"2105125582","DOI":"10.1093/intimm/dxn074","CorpusId":"1354343","PubMed":"18611945"},"title":"Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction.","abstract":"Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV."}},"tag":"EXP"},{"id":308,"details":{"paperId":"d859f0aa423aad4abbeee3a7cb078abe1664b714","externalIds":{"MAG":"2133134586","DOI":"10.1128/JVI.01813-10","CorpusId":"7097869","PubMed":"21307202"},"title":"Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer","abstract":"ABSTRACT The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni+2 column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming “hat pin”-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0"}},"tag":"EXP"},{"id":23,"details":{"paperId":"876353f51b5c3409a763ee9862747466e87bf2da","externalIds":{"MAG":"2043021763","DOI":"10.1006/VIRO.2002.1540","CorpusId":"25372054","PubMed":"12350355"},"title":"Sorting of the respiratory syncytial virus matrix protein into detergent-resistant structures is dependent on cell-surface expression of the glycoproteins.","abstract":"The interaction of the respiratory syncytial virus (RSV) Matrix (M) protein with the plasma membrane was investigated using polyclonal and monoclonal antisera raised against recombinant M expressed in bacteria. M bound mainly to the plasma membrane, although a significant proportion bound to internal membranes. However, no localisation of M with the Golgi was observed, suggesting that transport of M to the plasma membrane was independent of the transport mechanism for the viral glycoproteins. Expression from a recombinant baculovirus demonstrated the ability of M to bind membranes in the absence of viral glycoprotein expression. When cell-surface expression of the viral glycoproteins was prevented using Brefeldin A, M was still found in association with the plasma membrane, but the characteristics of M's membrane-binding ability were different to that found in untreated infected cells. In the presence of normal glycoprotein expression, M was sorted into lipid rafts and, in addition, formed structures that could only be disrupted by treatment with high salt buffers, a feature suggesting an interaction with the cytoskeleton or the formation of strong intramolecular associations. Brefeldin A prevented M from being sorted into lipid rafts or from forming strong intramolecular associations. Brefeldin A also affected the stability of M bound to the plasma membrane, as M was more readily dissociated in the presence of the inhibitor. Coexpression of M and F resulted in the incorporation of M into lipid rafts but did not cause the formation of the strong intramolecular bonds, suggesting that additional factors are required for this phenomena.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The interaction of the respiratory syncytial virus (RSV) Matrix (M) protein with the plasma membrane was investigated using polyclonal and monoclonal antisera raised against recombinant M expressed in bacteria, suggesting an interaction with the cytoskeleton or the formation of strong intramolecular associations."}},"tag":"EXP"},{"id":418,"details":{"paperId":"a8420d04644b05bc2b9d07d9fed5e0183344662c","externalIds":{"MAG":"2088304077","DOI":"10.1165/RCMB.2003-0424OC","CorpusId":"34994454","PubMed":"14742298"},"title":"Sphingosine kinase mediates activation of extracellular signal-related kinase and Akt by respiratory syncytial virus.","abstract":"Respiratory syncytial virus (RSV) preferentially infects lung epithelial cells. Infected cells remain viable well into the infection. This prolonged survival results from RSV-induced activation of pro-survival pathways, including Akt and extracellular signal-related kinase (ERK). Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite with demonstrated links to cell survival. It is enzymatically generated by sequential activation of ceramidase (generation of sphingosine) and sphingosine kinase (generation of S1P). In these studies, we found that RSV stimulated neutral ceramidase and sphingosine kinase activities in lung epithelial cells. The combined effect of activation of these two enzymes would decrease proapoptotic ceramide and increase antiapoptotic S1P. S1P activated Akt and ERK within minutes, and inhibition of sphingosine kinase blocked RSV-induced ERK and Akt activation, leading to accelerated cell death after viral infection. RSV infection does eventually kill infected cells but activation of cell survival pathways significantly delays cell death. The studies are the first evidence linking sphingolipid metabolites to cell survival mechanisms in the context of a viral infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"These studies are the first evidence linking sphingolipid metabolites to cell survival mechanisms in the context of a viral infection, and S1P activated Akt and ERK within minutes, and inhibition of sphingosine kinase blocked RSV-induced ERK and Akt activation, leading to accelerated cell death after viral infection."}},"tag":"EXP"},{"id":123,"details":{"paperId":"5443869dbe5aff104cb8749996bc0c5608adeabd","externalIds":{"MAG":"2900396794","DOI":"10.1021/acs.jmedchem.8b01361","CorpusId":"53243639","PubMed":"30411898"},"title":"State of the Art in Respiratory Syncytial Virus Drug Discovery and Development.","abstract":"Respiratory syncytial virus (RSV) is a globally prevalent viral infection with limited treatment options which hospitalizes millions each year. Treatment options have been limited to palivizumab, a monoclonal antibody, approved for prophylaxis in high-risk infants and ribavirin with very limited efficacy and significant safety concerns. This Perspective surveys the range of direct acting antiviral agents (DAAs) that target key steps in the viral life cycle. A number of approaches to DAAs have produced landmark clinical studies over the past few years, notably in fusion and nucleoside inhibitors, and an update of the clinical status of these compounds is provided. Non-nucleoside inhibitors of replication are reviewed in addition to inhibitors of other mechanisms, notably the RSV N and G proteins. This article will provide an informative perspective of the current status of drug discovery targeted at providing an effective therapy for RSV infection.","publicationTypes":["JournalArticle","Review"],"tldr":{"model":"tldr@v2.0.0","text":"An informative perspective of the current status of drug discovery targeted at providing an effective therapy for RSV infection is provided."}},"tag":"DRUG"},{"id":147,"details":{"paperId":"add7e78d3da89a677c7daac3c2ccbbf30c1b86a9","externalIds":{"PubMedCentral":"8556396","DOI":"10.1038/s42003-021-02754-2","CorpusId":"240229374","PubMed":"34716403"},"title":"Statin-mediated disruption of Rho GTPase prenylation and activity inhibits respiratory syncytial virus infection","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that statins inhibit post-entry events in the replicative cycle of RSV through a combination of cholesterol and isoprenoid-mediated effects, the latter involving a loss of Rho GTPase activity via disruption of protein prenylation."}},"tag":"EXP"},{"id":470,"details":{"paperId":"7c32769031304d524339802e3cc9cfe5f8025c75","externalIds":{"PubMedCentral":"3768923","MAG":"2122030533","DOI":"10.1590/S1517-83822011000100043","CorpusId":"8329659","PubMed":"24031640"},"title":"Structural Analysis of Human Respiratory Syncytial Virus P Protein: Identification of Intrinsically Disordered Domains","abstract":"Human Respiratory Syncytial Virus P protein plus the viral RNA, N and L viral proteins, constitute the viral replication complex. In this report we describe that HRSV P protein has putative intrinsically disordered domains predicted by in silico methods. These two domains, located at the amino and caboxi terminus, were identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria. The degradation is not occurring at the central oligomerization domain, since we also demonstrate that the purified fragments are able to oligomerize, similarly to the protein expressed in cells infected by HRSV. Disordered domains can play a role in protein interaction, and the present data contribute to the comprehension of HRSV P protein interactions in the viral replication complex.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is described that HRSV P protein has putative intrinsically disordered domains, located at the amino and caboxi terminus, identified by mass spectrometry analysis of peptides obtained from degradation fragments observed in purified P protein expressed in bacteria."}},"tag":"EXP"},{"id":275,"details":{"paperId":"8ee88deaa95a9ba1a1b34bce794cc64522b082f3","externalIds":{"MAG":"2111542813","DOI":"10.1128/JVI.00256-14","CorpusId":"22844459","PubMed":"24760890"},"title":"Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex","abstract":"ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCE hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Cryo-electron tomography studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization."}},"tag":"EXP"},{"id":493,"details":{"paperId":"a2c6ae9328d935208996bb1683df314bf6febff4","externalIds":{"PubMedCentral":"8658883","DOI":"10.3390/molecules26237398","CorpusId":"245011645","PubMed":"34885979"},"title":"Structural Characterization of Ectodomain G Protein of Respiratory Syncytial Virus and Its Interaction with Heparan Sulfate: Multi-Spectroscopic and In Silico Studies Elucidating Host-Pathogen Interactions","abstract":"The global burden of disease caused by a respiratory syncytial virus (RSV) is becoming more widely recognized in young children and adults. Heparan sulfate helps in attaching the virion through G protein with the host cell membrane. In this study, we examined the structural changes of ectodomain G protein (edG) in a wide pH range. The absorbance results revealed that protein maintains its tertiary structure at physiological and highly acidic and alkaline pH. However, visible aggregation of protein was observed in mild acidic pH. The intrinsic fluorescence study shows no significant change in the λmax except at pH 12.0. The ANS fluorescence of edG at pH 2.0 and 3.0 forms an acid-induced molten globule-like state. The denaturation transition curve monitored by fluorescence spectroscopy revealed that urea and GdmCl induced denaturation native (N) ↔ denatured (D) state follows a two-state process. The fluorescence quenching, molecular docking, and 50 ns simulation measurements suggested that heparan sulfate showed excellent binding affinity to edG. Our binding study provides a preliminary insight into the interaction of edG to the host cell membrane via heparan sulfate. This binding can be inhibited using experimental approaches at the molecular level leading to the prevention of effective host–pathogen interaction.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The binding study provides a preliminary insight into the interaction of edG to the host cell membrane via heparan sulfate, which can be inhibited using experimental approaches at the molecular level leading to the prevention of effective host–pathogen interaction."}},"tag":"EXP"},{"id":501,"details":{"paperId":"ba18bdddc0ecf67f5de08f769dd7f0122a1c26fd","externalIds":{"PubMedCentral":"8147935","DOI":"10.3390/v13050834","CorpusId":"235196562","PubMed":"34063087"},"title":"Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes","abstract":"RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (−) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5′-RNA capping and methylation, and 3′ end polyadenylation. Genome replication includes (+) RNA antigenome and (−) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"An overview of the RSV RNA synthesis and the structural insights into theRSV gene transcription and genome replication process are provided and a model of how the essential four proteins coordinate their activities in different RNA synthesis processes is proposed."}},"tag":"EXP"},{"id":366,"details":{"paperId":"b9c08006f100d9e51c0a494443078db03468103e","externalIds":{"MAG":"2145497813","DOI":"10.1128/JVI.74.21.9858-9867.2000","CorpusId":"28468870","PubMed":"11024112"},"title":"Structural Phosphoprotein M2-1 of the Human Respiratory Syncytial Virus Is an RNA Binding Protein","abstract":"ABSTRACT The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays. It is able to bind HRSV leader RNA specifically with cooperative kinetics, with an apparentKd of at least 90 nM. It also binds to long RNAs with no sequence specificity. The RNA binding domain has been located between amino acid residues 59 and 85, at the NH2terminus of the protein. This region contains the phosphorylatable amino acid residues threonine 56 and serine 58, whose modification decreases the binding capacity of M2-1 protein to long RNAs.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays."}},"tag":"EXP"},{"id":245,"details":{"paperId":"2dfddbcfb82d628b7217346f4f70f0ed12e4b710","externalIds":{"MAG":"2162323014","DOI":"10.1099/VIR.0.81430-0","CorpusId":"17887522","PubMed":"16361428"},"title":"Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an alpha-helical domain involved in oligomerization.","abstract":"Human respiratory syncytial virus (HRSV) phosphoprotein (P), an essential cofactor of the viral polymerase, is much shorter (241 aa) than and has no sequence similarity to P of other paramyxoviruses. Nevertheless, bioinformatic analysis of HRSV P sequence revealed a modular organization, reminiscent of other paramyxovirus Ps, with a central structured domain (aa 100-200), flanked by two intrinsically disordered regions (1-99 and 201-241). To test the predicted structure experimentally, HRSV P was purified from cell extracts infected with recombinant vaccinia virus or HRSV. The estimated molecular mass of P by gel filtration (approximately 500 kDa) greatly exceeded the theoretical mass of a homotetramer, proposed as the oligomeric form of native P. Nevertheless, the profile of cross-linked products obtained with purified P resembled that reported by others with P purified from bacteria or mammalian cells. Thus, the shape of HRSV P probably influences its elution from the gel filtration column, as reported for other paramyxovirus Ps. Digestion of purified HRSV P with different proteases identified a trypsin-resistant fragment (X) that reacted with a previously characterized monoclonal antibody (021/2P). N-terminal sequencing and mass spectrometry analysis placed the X fragment boundaries (Glu-104 and Arg-163) within the predicted structured domain of P. Cross-linking and circular dichroism analyses indicated that fragment X was oligomeric, with a high alpha-helical content, properties resembling those of the multimerization domain of Sendai and rinderpest virus P. These results denote structural features shared by HRSV and other paramyxovirus Ps and should assist in elucidation of the HRSV P structure.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Binformatic analysis of HRSV P sequence revealed a modular organization, reminiscent of other paramyxovirus Ps, with a central structured domain, flanked by two intrinsically disordered regions, and a trypsin-resistant fragment (X) that reacted with a previously characterized monoclonal antibody."}},"tag":"EXP"},{"id":512,"details":{"paperId":"0ecc781e6b61f217a15baae23443b4e9aff96e7b","externalIds":{"MAG":"1572789065","DOI":"10.5772/28747","CorpusId":"43325401"},"title":"Structural and Functional Aspects of the Small Hydrophobic (SH) Protein in the Human Respiratory Syncytial Virus","abstract":"hRSV is the leading cause of respiratory disease in infants, elderly, and immunocompromised populations worldwide (Falsey et al., 2005; Nair et al., 2010). Most individuals are infected at a young age, before 3 years old (Glezen et al., 1986). In fact, RSV infection is the most common cause of hospitalization in children 5 years old and below. When severe infection occurs, respiratory airways and pulmonary development are affected. However, the viral determinants of disease severity are not well defined, as little is known about its molecular mechanism of pathogenesis.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The most common cause of hospitalization in children 5 years old and below is RSV infection, and the viral determinants of disease severity are not well defined."}},"tag":"EXP"},{"id":161,"details":{"paperId":"a87390bd60bccd74e68527ffde729569222ab9ef","externalIds":{"DOI":"10.1073/pnas.2020587118","CorpusId":"232090233","PubMed":"33649232"},"title":"Structural basis for IFN antagonism by human respiratory syncytial virus nonstructural protein 2","abstract":"Significance The human respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract infections in the young and the elderly and a substantial burden to global human health. Currently, there are no specific and effective treatments for RSV infections. Here we report the first X-ray crystal structure of RSV nonstructural protein 2 (NS2), which revealed a unique fold. The combined biochemical and structural analyses of the NS2 structure identified a region that binds to and inhibits ubiquitination of an inactive form of RIG-I and MDA5, preventing downstream signaling and type I interferon production. Our study provides structural insight into the mechanism of how NS2 antagonizes IFN response to RSV infection and defines a target for antiviral development. Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I–like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNβ messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The first X-ray crystal structure of RSV nonstructural protein 2 (NS2) is reported, which revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1."}},"tag":"EXP"},{"id":137,"details":{"paperId":"ce854040ccf24a489bc92d16b24f04536b4d5595","externalIds":{"MAG":"2729120756","DOI":"10.1038/nmicrobiol.2017.101","CorpusId":"13480170","PubMed":"28665409"},"title":"Structural basis for human respiratory syncytial virus NS1-mediated modulation of host responses","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The crystal structure of hRSV NS1 protein is reported, which suggests that NS1 is a structural paralogue of h RSV matrix (M) protein, which provides a framework to target NS1 for therapeutic development to limit hRSv-associated morbidity and mortality."}},"tag":"EXP"},{"id":163,"details":{"paperId":"5df48f143cebab64a184d23d1a73dc5ab5a8d0af","externalIds":{"MAG":"2045472151","DOI":"10.1073/PNAS.260499197","CorpusId":"40439663","PubMed":"11106388"},"title":"Structural characterization of the human respiratory syncytial virus fusion protein core.","abstract":"Human respiratory syncytial virus (HRSV) is a major cause of a number of severe respiratory diseases, including bronchiolitis and pneumonia, in infants and young children. The HRSV F protein, a glycoprotein essential for viral entry, is a primary target for vaccine and drug development. Two heptad-repeat regions within the HRSV F sequence were predicted by the computer program learncoil-vmf. These regions are thought to form trimer-of-hairpins-like structures, similar to those found in the fusion proteins of several enveloped viruses. The hairpin structure likely brings the viral and cellular membranes into close apposition, thereby facilitating membrane fusion and subsequent viral entry. Here, we show that peptides, denoted HR-N and HR-C, corresponding to the heptad-repeat regions from the N-terminal and C-terminal segments of the HRSV F protein, respectively, form a stable alpha-helical trimer of heterodimers. The HRSV N/C complex was crystallized and its x-ray structure was determined at 2.3-A resolution. As anticipated, the complex is a six-helix bundle in which the HR-N peptides form a three-stranded, central coiled coil, and the HR-C peptides pack in an antiparallel manner into hydrophobic grooves on the coiled-coil surface. There is remarkable structural similarity between the HRSV N/C complex and the fusion protein core of other viruses, including HIV-1 gp41. In addition, earlier work has shown that HRSV HR-C peptides, like the HIV-1 gp41 C peptides, inhibit viral infection. Thus, drug discovery and vaccine development strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of HRSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that peptides, denoted HR-N and HR-C, corresponding to the heptad-repeat regions from the N and C-terminal segments of the HRSV F protein, respectively, form a stable alpha-helical trimer of heterodimers, similar to those found in the fusion proteins of several enveloped viruses."}},"tag":"EXP"},{"id":5,"details":{"paperId":"b20c1dbff2a8f0a5f4cdc657b3b7e267d91de112","externalIds":{"MAG":"2126674362","DOI":"10.1002/prot.21988","CorpusId":"33028867","PubMed":"18300250"},"title":"Structural properties of the human respiratory syncytial virus P protein: Evidence for an elongated homotetrameric molecule that is the smallest orthologue within the family of paramyxovirus polymerase cofactors","abstract":"The oligomeric state and the hydrodynamic properties of human respiratory syncytial virus (HRSV) phosphoprotein (P), a known cofactor of the viral RNA‐dependent RNA polymerase (L), and a trypsin‐resistant fragment (X) that includes its oligomerization domain were analyzed by sedimentation equilibrium and velocity using analytical ultracentrifugation. The results obtained demonstrate that both P and fragment X are homotetrameric with elongated shapes, consistent with electron micrographs of the purified P protein in which thin rod‐like molecules of ∼12.5 ± 1.0 nm in length were observed. A new chymotrypsin resistant fragment (Y*) included in fragment X has been identified and purified by gel filtration chromatography. Fragment Y* may represent a minimal version of the P oligomerization domain. Thermal denaturation curves based on circular dichroism data of P protein showed a complex behavior. In contrast, melting data generated for fragments X and particularly fragment Y* showed more homogeneous transitions indicative of simpler structures. A three‐dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data. This model will be an useful tool to make rational mutations and test the role of specific amino acids in the oligomerization and functional properties of the HRSV P protein. Proteins 2008. © 2008 Wiley‐Liss, Inc.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A three‐dimensional model of X and Y* fragments was built based on the atomic structure of the P oligomerization domain of the related Sendai virus, which is in good agreement with the experimental data."}},"tag":"EXP"},{"id":29,"details":{"paperId":"6871637406ef280f3ef404ac358d88e7798e3f31","externalIds":{"MAG":"2808498751","DOI":"10.1007/978-981-10-8456-0_11","CorpusId":"49193914","PubMed":"29900500"},"title":"Structure and Function of the Human Respiratory Syncytial Virus M2-1 Protein.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"An overview of the current understanding of the structure and function of HRSV M2-1 is presented, drawing on additional information derived from its structural homologues from other related viruses."}},"tag":"EXP"},{"id":444,"details":{"paperId":"a34e0d5b9985f10802ace6b7337b286b898725ee","externalIds":{"PubMedCentral":"3364950","MAG":"2033434959","DOI":"10.1371/journal.ppat.1002734","CorpusId":"1851697","PubMed":"22675274"},"title":"Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein","abstract":"Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The NMR structure of the RSV M2-158–177 core domain is reported, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M1-1."}},"tag":"EXP"},{"id":334,"details":{"paperId":"99e8bd40c7ba445e2042515ec40d08ac1d13df55","externalIds":{"MAG":"1583974749","DOI":"10.1128/jvi.63.11.4767-4776.1989","CorpusId":"22176194","PubMed":"2677404"},"title":"Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation","abstract":"The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation."}},"tag":"EXP"},{"id":28,"details":{"paperId":"0710bf28a6b7ca22741fc6ced46e5138133c97ad","externalIds":{"MAG":"2151727763","DOI":"10.1007/978-3-642-38919-1_4","CorpusId":"12412504","PubMed":"24362685"},"title":"Structure and function of respiratory syncytial virus surface glycoproteins.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The structure and function of the RSV surface glycoproteins are reviewed, including recent X-ray crystallographic data of the F glycoprotein in its pre- and postfusion conformations, and how this information informs antigen selection and vaccine development is discussed."}},"tag":"EXP"},{"id":39,"details":{"paperId":"e02c1bf0b1dc78b68d2213f73506be5400d2dc99","externalIds":{"MAG":"2547858930","DOI":"10.1007/s00894-016-3150-6","CorpusId":"19807145","PubMed":"27817112"},"title":"Structure and functional dynamics characterization of the ion channel of the human respiratory syncytial virus (hRSV) small hydrophobic protein (SH) transmembrane domain by combining molecular dynamics with excited normal modes","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This protocol can support future investigations of drug candidates by acting on SH protein to inhibit viral infection by investigating functional conformational changes of transmembrane proteins that act as molecular channels."}},"tag":"EXP"},{"id":260,"details":{"paperId":"8a7b5fdcd42aa5ab5d9170f2384aa32d2ce50a71","externalIds":{"MAG":"1985678343","DOI":"10.1110/ps.073366208","CorpusId":"45919220","PubMed":"18369195"},"title":"Structure and ion channel activity of the human respiratory syncytial virus (hRSV) small hydrophobic protein transmembrane domain","abstract":"The small hydrophobic (SH) protein from the human respiratory syncytial virus (hRSV) is a glycoprotein of ∼64 amino acids with one putative α‐helical transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. Herein, we have studied the secondary structure, orientation, and oligomerization of the transmembrane domain of SH (SH‐TM) in the presence of lipid bilayers. Only one oligomer, a pentamer, was observed in PFO‐PAGE. Using polarized attenuated total reflection‐Fourier transform infrared (PATR‐FTIR) spectroscopy, we show that the SH‐TM is α‐helical. The rotational orientation of SH‐TM was determined by site‐specific infrared dichroism (SSID) at two consecutive isotopically labeled residues. This orientation is consistent with that of an evolutionary conserved pentameric model obtained from a global search protocol using 13 homologous sequences of RSV. Conductance studies of SH‐TM indicate ion channel activity, which is cation selective, and inactive below the predicted pKa of histidine. Thus, our results provide experimental evidence that the transmembrane domain of SH protein forms pentameric α‐helical bundles that form cation‐selective ion channels in planar lipid bilayers. We provide a model for this pore, which should be useful in mutagenesis studies to elucidate its role during the virus cycle.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Experimental evidence is provided that the transmembrane domain of SH protein forms pentameric α‐helical bundles that form cation‐selective ion channels in planar lipid bilayers, which is cation selective, and inactive below the predicted pKa of histidine."}},"tag":"EXP"},{"id":259,"details":{"paperId":"02ccd5ecceffb5781beb166e16fe58b9be19a24c","externalIds":{"PubMedCentral":"5947689","MAG":"2775510829","DOI":"10.1107/S2053230X17017381","CorpusId":"206367192","PubMed":"29372904"},"title":"Structure and stability of the Human respiratory syncytial virus M2–1 RNA-binding core domain reveals a compact and cooperative folding unit","abstract":"A high-resolution crystal structure of the M2–1 RNA-binding domain of Human syncytial respiratory virus was determined. A combination of crystallography and SAXS indicated a role in its C-terminal extension.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A high-resolution crystal structure of the M2–1 RNA-binding domain of Human syncytial respiratory virus was determined and a combination of crystallography and SAXS indicated a role in its C-terminal extension."}},"tag":"EXP"},{"id":59,"details":{"paperId":"d6b3816f07af12dd738e891145158459b56ed6e2","externalIds":{"MAG":"2971906485","PubMedCentral":"7111336","DOI":"10.1016/j.cell.2019.08.014","CorpusId":"201835064","PubMed":"31495574"},"title":"Structure of the Respiratory Syncytial Virus Polymerase Complex","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The 3.2-Å cryo-EM structure of RSV L bound to tetrameric P reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation, which provides a framework for determining the molecular underpinnings ofRSV replication and transcription and should facilitate the design of effective RSV inhibitors."}},"tag":"EXP"},{"id":258,"details":{"paperId":"82cc7aa011fad77c1532e01518289be2655f3c90","externalIds":{"MAG":"2021688512","DOI":"10.1107/S1744309111029228","CorpusId":"10413444","PubMed":"22102022"},"title":"Structures of respiratory syncytial virus nucleocapsid protein from two crystal forms: details of potential packing interactions in the native helical form.","abstract":"Respiratory syncytial virus (RSV) is a frequent cause of respiratory illness in infants, but there is currently no vaccine nor effective drug treatment against this virus. The RSV RNA genome is encapsidated and protected by a nucleocapsid protein; this RNA-nucleocapsid complex serves as a template for viral replication. Interest in the nucleocapsid protein has increased owing to its recent identification as the target site for novel anti-RSV compounds. The crystal structure of human respiratory syncytial virus nucleocapsid (HRSVN) was determined to 3.6 Å resolution from two crystal forms belonging to space groups P2(1)2(1)2(1) and P1, with one and four decameric rings per asymmetric unit, respectively. In contrast to a previous structure of HRSVN, the addition of phosphoprotein was not required to obtain diffraction-quality crystals. The HRSVN structures reported here, although similar to the recently published structure, present different molecular packing which may have some biological implications. The positions of the monomers are slightly shifted in the decamer, confirming the adaptability of the ring structure. The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The details of the inter-ring contacts in one crystal form revealed here suggest a basis for helical packing and that the stabilization of native HRSVN is via mainly ionic interactions."}},"tag":"EXP"},{"id":488,"details":{"paperId":"6d55aaa747fbeaf119753f2dcae6c5952f312f66","externalIds":{"MAG":"2985171249","PubMedCentral":"6912631","DOI":"10.3390/cells8111417","CorpusId":"207961911","PubMed":"31717900"},"title":"Subversion of Host Cell Mitochondria by RSV to Favor Virus Production is Dependent on Inhibition of Mitochondrial Complex I and ROS Generation","abstract":"Respiratory syncytial virus (RSV) is a key cause of severe respiratory infection in infants, immunosuppressed adults, and the elderly worldwide, but there is no licensed vaccine or effective, widely-available antiviral therapeutic. We recently reported staged redistribution of host cell mitochondria in RSV infected cells, which results in compromised respiratory activities and increased reactive oxygen species (ROS) generation. Here, bioenergetic measurements, mitochondrial redox-sensitive dye, and high-resolution quantitative imaging were performed, revealing for the first time that mitochondrial complex I is key to this effect on the host cell, whereby mitochondrial complex I subunit knock-out (KO) cells, with markedly decreased mitochondrial respiration, show elevated levels of RSV infectious virus production compared to wild-type cells or KO cells with re-expressed complex I subunits. This effect correlates strongly with elevated ROS generation in the KO cells compared to wild-type cells or retrovirus-rescued KO cells re-expressing complex I subunits. Strikingly, blocking mitochondrial ROS levels using the mitochondrial ROS scavenger, mitoquinone mesylate (MitoQ), inhibits RSV virus production, even in the KO cells. The results highlight RSV’s unique ability to usurp host cell mitochondrial ROS to facilitate viral infection and reinforce the idea of MitoQ as a potential therapeutic for RSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0"}},"tag":"EXP"},{"id":388,"details":{"paperId":"eef13988c1600bd75714622d0c595db35e844bb7","externalIds":{"MAG":"2104898251","DOI":"10.1128/JVI.78.8.4363-4369.2004","CorpusId":"86619307"},"title":"Suppression of the Induction of Alpha, Beta, and Gamma Interferons by the NS1 and NS2 Proteins of Human Respiratory Syncytial Virus in Human Epithelial Cells and Macrophages","abstract":"ABSTRACT Wild-type human respiratory syncytial virus (HRSV) is a poor inducer of alpha/beta interferons (IFN-α/β). However, recombinant HRSV lacking the NS1 and NS2 genes (ΔNS1/2) induced high levels of IFN-α and -β in human pulmonary epithelial cells (A549) as well as in macrophages derived from primary human peripheral blood monocytes. Results with NS1 and NS2 single- and double-gene-deletion viruses indicated that the two proteins function independently as well as coordinately to achieve the full inhibitory effect, with NS1 having a greater independent role. The relative contributions of the individual NS proteins were the converse of that recently described for bovine RSV (J. F. Valarcher, J. Furze, S. Wyld, R. Cook, K. K. Conzelmann, and G. Taylor, J. Virol. 77:8426-8439, 2003). This pattern of inhibition by HRSV NS1 and NS2 also extended to the newly described antiviral cytokines IFN-λ1, -2 and -3.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Results with HRSV NS1 and NS2 single- and double-gene-deletion viruses indicated that the two proteins function independently as well as coordinately to achieve the full inhibitory effect, with NS1 having a greater independent role."}},"tag":"EXP"},{"id":159,"details":{"paperId":"1e1f4b21cf4b4febe87ad45d0ff5837a5ed970a5","externalIds":{"MAG":"1966142422","DOI":"10.1073/pnas.1400760111","CorpusId":"24658603","PubMed":"25056968"},"title":"Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells","abstract":"Significance Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in children worldwide. The induction of poor T-cell immunological memory causes a high susceptibility to reinfections, which contributes to hRSV spread. Previously, we showed that hRSV inhibits T-cell activation by impairing the assembly of the dendritic cell (DC)−T-cell immunological synapse (IS). Here, we show that the nucleoprotein (N) of hRSV—a canonical cytosolic protein—is expressed on the surface of infected DCs. Further, using the supported-lipid-bilayer system (that mimics the DC/ antigen-presenting cells-membrane composition), we observed that the hRSV N interfered with pMHC−T-cell receptor interactions and inhibited IS assembly. We conclude that hRSV N may therefore be instrumental in impairing the host immune response during infection with this virus. Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4+ T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell−bilayer interface, suggesting that N protein interferes with pMHC−TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"HRSV N protein expression at the surface of infected cells inhibits T-cell activation and is defined as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV."}},"tag":"EXP"},{"id":190,"details":{"paperId":"a3c07aa03cab73ba60beaadfa8c1dc929cff5e6c","externalIds":{"MAG":"2061029252","DOI":"10.1086/315134","CorpusId":"15171043","PubMed":"10558959"},"title":"Surfactant protein A binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity.","abstract":"Collectins are a family of calcium-dependent collagenous lectins that appear to be important in innate host defense. We investigated the ability of three human collectins, namely, lung surfactant proteins A (SP-A) and D (SP-D) and the serum mannose-binding protein (MBP), to bind to the surface glycoproteins of respiratory syncytial virus (RSV). SP-A was shown to bind to the F (fusion) glycoprotein but not to the viral G (attachment) glycoprotein, and binding was completely abrogated in the presence of EDTA. Neither SP-D nor MBP bound to either glycoprotein. SP-A also neutralized RSV in a calcium dependent fashion. These results support a role for SP-A in the defense of infants against infection with RSV and indicate a possible mechanism for its protective activity.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results support a role for SP-A in the defense of infants against infection with RSV and indicate a possible mechanism for its protective activity."}},"tag":"EXP"},{"id":252,"details":{"paperId":"735ebc33d739fbc16bd427f3f3a88028e7f8c13e","externalIds":{"DOI":"10.1101/2021.03.09.434545","CorpusId":"232223842"},"title":"Synchronization Temperature Determines the Location of RSV Fusion During Entry in Cultured Cells","abstract":"Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in children under five years of age. No vaccines against this virus are currently available. RSV infection of a cell is initiated by fusion between the virion membrane and a cellular membrane, but it is not clear if the fusion process takes place at the plasma membrane or within an endosome. Most such experiments have been initiated at the traditional synchronization temperature of 4°C, an abnormal temperature for animal cells and one at which cellular homeostasis may be negatively affected. We have compared two synchronization temperatures (4°C and 22°C) to determine the kinetics of RSV entry into human bronchial epithelial cells. Following inoculation, virus entry was halted at different times by the addition of neutralizing antibody or temperature reduction to 4°C. We engineered a virus that encodes an extra viral gene, beta-lactamase fused to the viral phosphoprotein (P), to enable rapid detection after infection initiation. We found that the synchronization temperature used during inoculation determines the site of fusion. Transition from 4°C to 37°C resulted in RSV entry via the endosomal pathway but also induced F-actin disruption and plasma membrane blebbing, whether the cells were inoculated with RSV or not. Transition from 22°C to 37°C resulted in RSV entry by fusion at the plasma membrane and without the F-actin and plasma membrane disruptions. These results suggest that RSV normally enters cells by fusion at the plasma membrane and that the induction of endocytosis by infection synchronization at 4°C may be an artefact caused by distortion of the plasma membrane-supporting cytoskeleton. Author Summary In order to understand the overall mechanism driving infection, it is important to determine how the virus enters cells. The pathway that RSV uses to infect cells is unclear. It is a common practice to attach the virions at 4°C to synchronize the viral infection. In this report, we found that warming up primary cultures of undifferentiated normal human bronchial epithelial cells to 37°C from 4°C triggered dramatic changes in their cell membrane and cytoskeleton totally unrelated to the presence of the virions. The assessment of viral content delivery to the cytoplasm using RSV engineered to express BlaM allowed us to find that the virions attached at 4°C or 22°C fused their envelope with endosome or plasma membrane, respectively. Consequently, the entry via endosome after attachment at 4°C is an experimental artefact and RSV infects by fusing its envelope with the plasma membrane. The implications go beyond RSV since the entry of several virus species have been explored by synchronizing the infection after attachment at 4°C.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"Warming up primary cultures of undifferentiated normal human bronchial epithelial cells to 37°C from 4°C triggered dramatic changes in their cell membrane and cytoskeleton totally unrelated to the presence of the virions, suggesting that RSV normally enters cells by fusion at the plasma membrane and that the induction of endocytosis by infection synchronization at 4 °C may be an artefact caused by distortion of the plasma membranes-supporting cytos skeleton."}},"tag":"EXP"},{"id":19,"details":{"paperId":"51008bf7032a9598a7a702850ad3aa35413e5654","externalIds":{"MAG":"2056072684","DOI":"10.1006/VIRO.2001.1105","CorpusId":"19382474","PubMed":"11601901"},"title":"Synergistic effects of gene-end signal mutations and the M2-1 protein on transcription termination by respiratory syncytial virus.","abstract":"Individual mononegavirus genes terminate with a short cis-acting element, the gene-end (GE) signal, that directs polyadenylation and termination and might also influence the efficiency of reinitiation at the next downstream gene. The 12-13 nucleotide (nt) GE signals of human respiratory syncytial virus (RSV) consist of a conserved pentanucleotide (3'-UCAAU, negative sense), followed by a 3-nt middle region that is AU-rich but otherwise not conserved, followed by a 4- or 5-nt poly(U) region that is thought to generate the poly(A) tail of the encoded mRNA by reiterative copying. Most of the naturally occurring differences in the GE signals of the various RSV genes occur in the \"middle\" and \"poly(U)\" regions. We mutated a copy of the fusion protein (F) GE signal that was positioned at the end of the promoter-proximal gene of a tricistronic minigenome and evaluated the effect of these mutations on RSV transcription in a plasmid-initiated, intracellular assay. Mutations confirmed the importance of the middle region's AU-rich nature and 3-nt length, and the poly(U) tract's 4-nt minimum functional length, with maximal termination efficiency observed at five U residues. Nt assignments other than U at position 13 also affected the efficiency of termination, showing that this position is part of the functional 13-nt GE signal. These results indicate that differences in nt assignments in the middle and poly(U) regions of the GE signal, which occur frequently in nature, affect the efficiency of termination. Unexpectedly, the ability of certain mutations to inhibit termination was completely dependent on coexpression of the M2-1 protein, and in many other cases the inhibitory effect of the mutation was greatly enhanced in the presence of M2-1. Thus, M2-1 appears to have the effect of altering the polymerase such that it ignores suboptimal GE signals. Interestingly, certain mutations that greatly decreased the efficiency of termination in the absence of M2-1 did not have much effect on the expression of the second gene, implying that correct termination and/or polyadenylation at the upstream gene is not obligatory for reinitiation at the next downstream gene.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Interestingly, M2-1 appears to have the effect of altering the polymerase such that it ignores suboptimal GE signals, implying that correct termination and/or polyadenylation at the upstream gene is not obligatory for reinitiation at the next downstream gene."}},"tag":"EXP"},{"id":405,"details":{"paperId":"08f9355ffe077de3a65ddc680cff4e43eac1f475","externalIds":{"MAG":"3097343917","PubMedCentral":"7803913","DOI":"10.1136/thoraxjnl-2020-215171","CorpusId":"225100502","PubMed":"33109690"},"title":"TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection","abstract":"Introduction Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%–40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required. Methods It is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl−) channel modulators to investigate their role during the HRSV life-cycle. Results We demonstrate the requirement for TMEM16A, a calcium-activated Cl− channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01. Discussion These findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The requirement for TMEM16A, a calcium-activated Cl− channel, for HRSV infection is demonstrated and revealed as an exciting target for future host-directed antiviral therapeutics."}},"tag":"EXP"},{"id":148,"details":{"paperId":"6e9bfabd6e13cb67e1d10ff5c5d321334b3f1a3f","externalIds":{"MAG":"2399023107","PubMedCentral":"4872165","DOI":"10.1038/srep25806","CorpusId":"4300569","PubMed":"27194388"},"title":"Targeting human respiratory syncytial virus transcription anti-termination factor M2-1 to inhibit in vivo viral replication","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication and opens a novel avenue for the development of future therapies against h RSV infection."}},"tag":"EXP"},{"id":312,"details":{"paperId":"8749564c4567091f409f2c470252c67096205e81","externalIds":{"DOI":"10.1128/JVI.02217-20","CorpusId":"230819460","PubMed":"33408180"},"title":"Tetramerization of Phosphoprotein Is Essential for Respiratory Syncytial Virus Budding while Its N-Terminal Region Mediates Direct Interactions with the Matrix Protein","abstract":"Human respiratory syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and antiviral drugs. ABSTRACT It was shown previously that the matrix (M), phosphoprotein (P), and fusion (F) proteins of respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells by using a split Nano luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using nuclear magnetic resonance (NMR), we identify three novel M interaction sites on P, namely, site I in the αN2 region, site II in the 115 to 125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs. IMPORTANCE Human respiratory syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and antiviral drugs. The lack of knowledge of the RSV budding mechanism presents a continuing challenge for virus-like particle (VLP) production for vaccine purposes. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of the RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that direct interaction between P and M modulates RSV VLP budding, which emphasizes P as a central regulator of the RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production."}},"tag":"EXP"},{"id":335,"details":{"paperId":"d879cce213384cacfd1578503ae42540a8871780","externalIds":{"MAG":"1637699647","DOI":"10.1128/jvi.63.5.2019-2029.1989","CorpusId":"43807763","PubMed":"2649692"},"title":"The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species","abstract":"The respiratory syncytial virus (RSV) 1A protein was previously identified as a 7.5-kilodalton (kDa) nonglycosylated species that, on the basis of its predicted sequence determined from the sequence of its mRNA, contains a hydrophobic central domain that was suggestive of membrane interaction. Here, four major, structurally distinct intracellular species of the 1A protein were identified in cells infected by RSV or by a recombinant vaccinia virus expressing the 1A gene. The four species of 1A were: (i) the previously described, nonglycosylated 7.5-kDa species that appeared to be the full-length, unmodified 1A protein; (ii) a nonglycosylated 4.8-kDa species that was carboxy-coterminal with the 7.5-kDa species and might be generated by translational initiation at the second AUG in the sequence; (iii) a 13- to 15-kDa species that contained one or two N-linked carbohydrate side chains of the high-mannose type; and (iv) a 21- to 30-kDa glycosylated species that appeared to be generated from the 13- to 15-kDa species by further modification of the N-linked carbohydrate. All four forms of the 1A protein were synthesized and processed on intracellular membranes, and several lines of biochemical evidence showed that all four species were integral membrane proteins. Thus, the 1A protein is a third RSV integral membrane protein and is present as such in both glycosylated and nonglycosylated forms. With the use of antiserum raised against a synthetic peptide representing the C terminus of the 1A protein, indirect immunofluorescence showed that the 1A protein was expressed at the cell surface. Antibody-antigen complexes formed at the surface of intact infected cells were immunoprecipitated, showing that the 7.5-kDa, 13- to 15-kDa, and 21- to 30-kDa, but not the 4.8-kDa, species, were accessible to extracellular antibodies. Thus, the 1A protein is a candidate to be a viral surface antigen. The small size, gene map location integral membrane association, and cell surface expression of the 1A protein strongly suggested that it is a counterpart to the SH protein that has been described for simian virus type 5. We suggest that, in the future, the RSV 1A protein be given the same designation, namely, SH.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The small size, gene map location integral membrane association, and cell surface expression of the 1A protein strongly suggested that it is a counterpart to the SH protein that has been described for simian virus type 5."}},"tag":"EXP"},{"id":285,"details":{"paperId":"b24bdd24fc02e25892acbe7f8ebb07a5ca08ea39","externalIds":{"MAG":"1965685650","DOI":"10.1128/JVI.00526-07","CorpusId":"9235460","PubMed":"17567697"},"title":"The 24-Angstrom Structure of Respiratory Syncytial Virus Nucleocapsid Protein-RNA Decameric Rings","abstract":"ABSTRACT Respiratory syncytial virus (RSV), a nonsegmented, negative-sense RNA-containing virus, is a common cause of lower respiratory tract disease. Expression of RSV nucleocapsid protein (N) in insect cells using the baculovirus expression system leads to the formation of N-RNA complexes that are morphologically indistinguishable from viral nucleocapsids. When imaged in an electron microscope, three distinct types of structures were observed: tightly wound short-pitch helices, highly extended helices, and rings. Negative stain images of N-RNA rings were used to calculate a three-dimensional reconstruction at 24 Å resolution, revealing features similar to those observed in nucleocapsids from other viruses of the order Mononegavirales. The reconstructed N-RNA rings comprise 10 N monomers and have an external radius of 83 Å and an internal radius of 40 Å. Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities.","publicationTypes":["JournalArticle","Study"],"tldr":{"model":"tldr@v2.0.0","text":"Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities."}},"tag":"EXP"},{"id":473,"details":{"paperId":"c09ee717aa8079e7f0a3a88e063250a7a194b4d6","externalIds":{"PubMedCentral":"3204419","MAG":"2151022126","DOI":"10.2174/1874357901105010114","CorpusId":"5650305","PubMed":"22046209"},"title":"The Anti-Apoptotic Effect of Respiratory Syncytial Virus on Human Peripheral Blood Neutrophils is Mediated by a Monocyte Derived Soluble Factor","abstract":"Respiratory Syncytial Virus (RSV) causes annual epidemics of respiratory disease particularly affecting infants. The associated airway inflammation is characterized by an intense neutrophilia. This neutrophilic inflammation appears to be responsible for much of the pathology and symptoms. Previous work from our group had shown that there are factors within the airways of infants with RSV bronchiolitis that inhibit neutrophil apoptosis. This study was undertaken to determine if RSV can directly affect neutrophil survival. Neutrophils were isolated from citrated venous blood (collected from healthy adult volunteers) by discontinuous plasma: Percoll gradient centrifugation and, in some experiments, further purified by negative immunomagnetic bead selection. The effect of RSV on neutrophil survival was measured by Annexin V-PE /To-Pro-3 staining and by morphological changes, using Dif-Quick staining of cytospins. Inhibition of neutrophil apoptosis was observed in neutrophils isolated by standard plasma:Percoll gradient when exposed to RSV but not in ultra pure neutrophil preparations. Adding monocytes back to ultra purified preparations restored the effect. The inhibition of apoptosis was observed with both active and UV inactivated virus. The effect is dependent on a soluble factor and appears to be dependent on CD14 receptors on the monocytes.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Inhibition of neutrophil apoptosis was observed in neutrophils isolated by standard plasma: Percoll gradient when exposed to RSV but not in ultra pure neutrophIL preparations, and the effect is dependent on a soluble factor and appears to be dependent on CD14 receptors on the monocytes."}},"tag":"EXP"},{"id":227,"details":{"paperId":"b468ff49d93e02c0c594bc11d3e670c5d7982c75","externalIds":{"MAG":"2183051223","DOI":"10.1099/0022-1317-81-4-919","CorpusId":"26938501","PubMed":"10725417"},"title":"The C-terminal third of human respiratory syncytial virus attachment (G) protein is partially resistant to protease digestion and is glycosylated in a cell-type-specific manner.","abstract":"The soluble form of the human respiratory syncytial virus (HRSV) attachment protein (Gs) was purified from the supernatant of infected cell cultures by immunoaffinity chromatography. Digestion of Gs with proteases and Western blot analysis identified two fragments that were partially resistant to protease degradation. Reactivity with diagnostic monoclonal antibodies located these two fragments in the primary structure of the G molecule. The large fragment spanned the C-terminal third of the G protein whereas the small fragment represented the N-terminal half of the large fragment. Purification of Gs from infected cells (either HEp-2 or M6) followed by protease digestion located host-cell-dependent glycosylation of the G protein in the unique part of the large protease-resistant fragment. The use of HRSV mutants encoding truncated G proteins allowed us to place some of the host-cell-dependent glycosylation differences in a small segment of the G protein. Interestingly, cell-specific glycosylations in the C-terminal half of the large protease-resistant fragment influenced the expression of certain epitopes located in its N-terminal half. These results bear important implications for the three-dimensional structure of the G glycoprotein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Interestingly, cell-specific glycosylations in the C-terminal half of the large protease-resistant fragment influenced the expression of certain epitopes located in its N-terminals, which bear important implications for the three-dimensional structure of the G glycoprotein."}},"tag":"EXP"},{"id":401,"details":{"paperId":"2d818f805959b1f7577035078129d552db7f8a4d","externalIds":{"PubMedCentral":"8649777","DOI":"10.1128/mBio.03075-21","CorpusId":"244909582","PubMed":"34872355"},"title":"The Cellular and Viral circRNAome Induced by Respiratory Syncytial Virus Infection","abstract":"Noncoding RNAs (ncRNAs) demonstrate substantial roles in cell-virus interactions. Circular RNAs (circRNAs) are a newly identified class of ncRNAs that have gained increased attention recently. ABSTRACT Circular RNAs (circRNAs) are a new class of noncoding RNAs that have gained increased attention. DNA virus infections have been reported to induce modifications in cellular circRNA transcriptomes and express viral circRNAs. However, the identification and expression of cellular and viral circRNAs are unknown in the context of respiratory syncytial virus (RSV), a human RNA virus with no effective treatments or vaccines. Here, we report a comprehensive identification of the cellular and viral circRNAs induced by RSV infection in A549 cells with high-throughput sequencing. In total, 53,719 cellular circRNAs and 2,280 differentially expressed cellular circRNAs were identified. Trend analysis further identified three significant expression pattern clusters, which were related to the antiviral immune response according to gene enrichment analysis. Subsequent results showed that not only RSV infection but also poly(I·C) treatment and another RNA virus infection induced the upregulation of the top 10 circRNAs from the focused cluster. The top 10 circRNAs generally inhibit RSV replication in turn. Moreover, 1,254 viral circRNAs were identified by the same circRNA sequencing. The induced expression of viral circRNAs by RSV infection was found not only in A549 cells but also in HEp-2 cells. Additionally, we profiled the general characteristics of both cellular and viral circRNAs such as back-splicing signals, etc. Collectively, RSV infection induced the differential expression of cellular circRNAs, some of which affected RSV infection, and RSV also expressed viral circRNAs. Our study reveals novel layers of host-RSV interactions and identifies cellular or viral circRNAs that may be novel therapeutic targets or biomarkers. IMPORTANCE Noncoding RNAs (ncRNAs) demonstrate substantial roles in cell-virus interactions. Circular RNAs (circRNAs) are a newly identified class of ncRNAs that have gained increased attention recently. DNA virus infections have been reported to induce modifications in cellular circRNA transcriptomes and express viral circRNAs. However, the identification and expression of cellular and viral circRNAs are unknown in the context of respiratory syncytial virus (RSV), a human RNA virus with no effective treatments or vaccines. Here, we report a comprehensive identification of the cellular and viral circRNAs induced by RSV infection by high-throughput sequencing. We revealed that RSV infection induces the differential expression of cellular circRNAs, some of which affected RSV infection, and that RSV also expresses viral circRNAs. Our study reveals novel layers of host-RSV interactions and identifies cellular or viral circRNAs that may be novel therapeutic targets or biomarkers.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A comprehensive identification of the cellular and viral circRNAs induced by RSV infection in A549 cells with high-throughput sequencing reveals novel layers of host-RSV interactions and identifies cellular or viral circ RNAs that may be novel therapeutic targets or biomarkers."}},"tag":"EXP"},{"id":374,"details":{"paperId":"d581177e7f41f235718cf6b65c80dfcb8dcb2dd4","externalIds":{"MAG":"2128289360","DOI":"10.1128/JVI.76.12.6164-6171.2002","CorpusId":"11578161","PubMed":"12021350"},"title":"The Central Conserved Cystine Noose of the Attachment G Protein of Human Respiratory Syncytial Virus Is Not Required for Efficient Viral Infection In Vitro or In Vivo","abstract":"ABSTRACT The G glycoprotein of human respiratory syncytial virus (RSV) was identified previously as the viral attachment protein. Although we and others recently showed that G is not essential for replication in vitro, it does affect the efficiency of replication in a cell type-dependent fashion and is required for efficient replication in vivo. The ectodomain of G is composed of two heavily glycosylated domains with mucin-like characteristics that are separated by a short central region that is relatively devoid of glycosylation sites. This central region contains a 13-amino acid segment that is conserved in the same form among RSV isolates and is overlapped by a second segment containing four cysteine residues whose spacings are conserved in the same form and which create a cystine noose. The conserved nature of the cystine noose and flanking 13-amino acid segment suggested that this region likely was important for attachment activity. To test this hypothesis, we constructed recombinant RSVs from which the region containing the cysteine residues was deleted together with part or all of the conserved 13-amino acid segment. Surprisingly, each deletion had little or no effect on the intracellular synthesis and processing of the G protein, the kinetics or efficiency of virus replication in vitro, or sensitivity to neutralization by soluble heparin in vitro. In addition, neither deletion had any discernible effect on the ability of RSV to infect the upper respiratory tract of mice and both resulted in a 3- to 10-fold reduction in the lower respiratory tract. Thus, although the G protein is necessary for efficient virus replication in vivo, this activity does not require the central conserved cystine noose region.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Although the G protein is necessary for efficient virus replication in vivo, this activity does not require the central conserved cystine noose region."}},"tag":"EXP"},{"id":504,"details":{"paperId":"2fcd5f59d73b2f3e74d76ab62023c90c5dd573c0","externalIds":{"PubMedCentral":"5620547","MAG":"2732813268","DOI":"10.3390/vaccines5030016","CorpusId":"26066091","PubMed":"28671606"},"title":"The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection","abstract":"Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182–186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSVG protein function and targeting."}},"tag":"EXP"},{"id":363,"details":{"paperId":"94277fefb11fa9369a1cfe5fc2ad1619a09fcef0","externalIds":{"MAG":"2148275674","DOI":"10.1128/JVI.74.13.5911-5920.2000","CorpusId":"12161643","PubMed":"10846072"},"title":"The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil","abstract":"ABSTRACT Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K 3 ≈ 2.2 × 1011 M−2) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with β-sheet-like characteristics. Only a small increase in α-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999). Finally, HR3 forms weak α-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results of these studies support the idea that viral fusion proteins have a common core architecture, and the isolated HR1, HR2, and HR3 domains of RSV F support this idea."}},"tag":"EXP"},{"id":362,"details":{"paperId":"8cc50e2e7848efd3ca4b77401fd8dad824202b94","externalIds":{"MAG":"2139557585","DOI":"10.1128/JVI.74.13.5880-5885.2000","CorpusId":"44636474","PubMed":"10846068"},"title":"The Cys3-His1 Motif of the Respiratory Syncytial Virus M2-1 Protein Is Essential for Protein Function","abstract":"ABSTRACT The M2 gene of respiratory syncytial (RS) virus has two open reading frames (ORFs). ORF1 encodes a 22-kDa protein termed M2-1. The M2-1 protein contains a Cys3-His1 motif (C-X7-C-X5-C-X3-H) near the amino terminus. This motif is conserved in all human, bovine, and ovine strains of RS virus. A similar motif found in the mammalian transcription factor Nup475 has been shown to bind zinc. The M2-1 protein of human RS virus functions as a transcription factor which increases polymerase processivity, and it enhances readthrough of intergenic junctions during RS virus transcription, thereby acting as a transcription antiterminator. The M2-1 protein also interacts with the nucleocapsid protein. We examined the effects of mutations of cysteine and histidine residues predicted to coordinate zinc in the Cys3-His1 motif on transcription antitermination and N protein binding. We found that mutating the predicted zinc-coordinating residues, the cysteine residues at amino acid positions 7 and 15 and the histidine residue at position 25, prevented M2-1 from enhancing transcriptional readthrough. In contrast, mutations of amino acids within this motif not predicted to coordinate zinc had no effect. Mutations of the predicted zinc-coordinating residues in the Cys3-His1 motif also prevented M2-1 from interacting with the nucleocapsid protein. One mutation of a noncoordinating residue in the motif which did not affect readthrough during transcription, E10G, prevented interaction with the nucleocapsid protein. This suggests that M2-1 does not require interaction with the nucleocapsid protein in order to function during transcription. Analysis of the M2-1 protein in reducing sodium dodecyl sulfate-polyacrylamide gels revealed two major forms distinguished by their mobilities. The slower migrating form was shown to be phosphorylated, whereas the faster migrating form was not. Mutations in the Cys3-His1 motif caused a change in distribution of the M2-1 protein from the slower to the faster migrating form. The data presented here show that the Cys3-His1 motif of M2-1 is essential for maintaining the functional integrity of the protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data presented here show that the Cys3-His1 motif of M2-1 is essential for maintaining the functional integrity of the protein and does not require interaction with the nucleocapsid protein in order to function during transcription."}},"tag":"EXP"},{"id":318,"details":{"paperId":"78dd7f1c72fd7749596e62a8aca26578564ec66a","externalIds":{"MAG":"2116598331","DOI":"10.1128/JVI.02671-05","CorpusId":"31836126","PubMed":"16731924"},"title":"The Cysteine-Rich Region and Secreted Form of the Attachment G Glycoprotein of Respiratory Syncytial Virus Enhance the Cytotoxic T-Lymphocyte Response despite Lacking Major Histocompatibility Complex Class I-Restricted Epitopes","abstract":"ABSTRACT The cytotoxic T-lymphocyte (CTL) response is important for the control of viral replication during respiratory syncytial virus (RSV) infection. The attachment glycoprotein (G) of RSV does not encode major histocompatibility complex class I-restricted epitopes in BALB/c mice (H-2d). Furthermore, studies to date have described an absence of significant CTL activity directed against this protein in humans. Therefore, G previously was not considered necessary for the generation of RSV-specific CTL responses. In this study, we demonstrate that, despite lacking H-2d-restricted epitopes, G enhances the generation of an effective CTL response against RSV. Furthermore, we show that this stimulatory effect is independent of virus titers and RSV-induced inflammation; that it is associated primarily with the secreted form of G; and that the effect depends on the cysteine-rich region of G (GCRR), a segment conserved in wild-type isolates worldwide. These findings reveal a novel function for the GCRR with potential implications for the generation of protective cellular responses and vaccine development.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that, despite lacking H-2d-restricted epitopes, G enhances the generation of an effective CTL response against RSV and is associated primarily with the secreted form of G (GCRR), a segment conserved in wild-type isolates worldwide."}},"tag":"EXP"},{"id":302,"details":{"paperId":"1ab199e6586a3a2d6d746444e7967048a387ffe9","externalIds":{"MAG":"2126604662","DOI":"10.1128/JVI.01439-06","CorpusId":"641378","PubMed":"16928754"},"title":"The Cytoplasmic Tail of the Human Respiratory Syncytial Virus F Protein Plays Critical Roles in Cellular Localization of the F Protein and Infectious Progeny Production","abstract":"ABSTRACT The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction."}},"tag":"EXP"},{"id":439,"details":{"paperId":"9f980e823f7f4bacd674f8bf21efd169612cc5af","externalIds":{"PubMedCentral":"4257679","MAG":"1997702073","DOI":"10.1371/journal.pone.0114447","CorpusId":"2101801","PubMed":"25479059"},"title":"The Eukaryotic Elongation Factor 1A Is Critical for Genome Replication of the Paramyxovirus Respiratory Syncytial Virus","abstract":"The eukaryotic translation factor eEF1A assists replication of many RNA viruses by various mechanisms. Here we show that down-regulation of eEF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV) life cycle. The key proteins in the replicase/transcriptase complex of RSV; the nucleocapsid (N) protein, phosphoprotein (P) and matrix (M) protein, all associate with eEF1A in RSV infected cells, although N is the strongest binding partner. Using individually expressed proteins, N, but not P or M bound to eEF1A. This study demonstrates a novel interaction between eEF1A and the RSV replication complex, through binding to N protein, to facilitate genomic RNA synthesis and virus production.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that down-regulation of e EF1A restricts the expression of viral genomic RNA and the release of infectious virus, demonstrating a biological requirement for eEF1A in the respiratory syncytial virus (RSV) life cycle."}},"tag":"EXP"},{"id":365,"details":{"paperId":"7dd6bade530e7f0b81f686344351b8fe371abd54","externalIds":{"MAG":"2125818108","DOI":"10.1128/JVI.74.14.6442-6447.2000","CorpusId":"16149016","PubMed":"10864656"},"title":"The Fusion Glycoprotein of Human Respiratory Syncytial Virus Facilitates Virus Attachment and Infectivity via an Interaction with Cellular Heparan Sulfate","abstract":"ABSTRACT Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED50] = 0.28 ± 0.11 μg/ml) andcp-52 (ED50 = 0.55 ± 0.14 μg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity."}},"tag":"EXP"},{"id":309,"details":{"paperId":"92c197a104013a0050b6cfce498960ca32a9de5c","externalIds":{"MAG":"2155115423","DOI":"10.1128/JVI.01887-07","CorpusId":"16104411","PubMed":"18216092"},"title":"The Fusion Protein of Respiratory Syncytial Virus Triggers p53-Dependent Apoptosis","abstract":"ABSTRACT Infection with respiratory syncytial virus (RSV) frequently causes inflammation and obstruction of the small airways, leading to severe pulmonary disease in infants. We show here that the RSV fusion (F) protein, an integral membrane protein of the viral envelope, is a strong elicitor of apoptosis. Inducible expression of F protein in polarized epithelial cells triggered caspase-dependent cell death, resulting in rigorous extrusion of apoptotic cells from the cell monolayer and transient loss of epithelial integrity. A monoclonal antibody directed against F protein inhibited apoptosis and was also effective if administered to A549 lung epithelial cells postinfection. F protein expression in epithelial cells caused phosphorylation of tumor suppressor p53 at serine 15, activation of p53 transcriptional activity, and conformational activation of proapoptotic Bax. Stable expression of dominant-negative p53 or p53 knockdown by RNA interference inhibited the apoptosis of RSV-infected A549 cells. HEp-2 tumor cells with low levels of p53 were not sensitive to RSV-triggered apoptosis. We propose a new model of RSV disease with the F protein as an initiator of epithelial cell shedding, airway obstruction, secondary necrosis, and consequent inflammation. This makes the RSV F protein a key target for the development of effective postinfection therapies.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A new model of RSV disease is proposed with the F protein as an initiator of epithelial cell shedding, airway obstruction, secondary necrosis, and consequent inflammation, which makes the RSV F protein a key target for the development of effective postinfection therapies."}},"tag":"EXP"},{"id":378,"details":{"paperId":"6b62a31da6fc71e7d62f51f121612b2691978506","externalIds":{"MAG":"2127278256","DOI":"10.1128/JVI.77.11.6580-6584.2003","CorpusId":"36786206","PubMed":"12743318"},"title":"The G Glycoprotein of Respiratory Syncytial Virus Depresses Respiratory Rates through the CX3C Motif and Substance P","abstract":"ABSTRACT Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G Glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycop protein, anti-SP, or anti-C X3 CR1 monoclonal antibodies."}},"tag":"EXP"},{"id":215,"details":{"paperId":"9812b6bc5a56e3ceabc8ad498a13555393f90b22","externalIds":{"MAG":"2099242857","DOI":"10.1099/0022-1317-72-3-669","CorpusId":"22018727","PubMed":"2005433"},"title":"The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity.","abstract":"The reactivities of eighteen monoclonal antibodies with different glycosylated forms of the human respiratory syncytial (RS) virus G protein were tested in Western blots. Only five antibodies recognized the unglycosylated precursor. The majority of antibodies, however, reacted with the O-glycosylated form of the G protein, emphasizing the importance of this type of modification for the antigenicity of the mature molecule. Human antisera, which recognized the RS virus G protein in Western blots, failed to inhibit the binding of anti-G antibodies to the virus but inhibited the binding of anti-F antibodies in the same type of assay. The human antibodies, however, did not recognize the G protein of some neutralization-resistant mutants selected with one anti-G monoclonal antibody. These mutants contain drastic amino acid sequence changes in the C-terminal end of the G molecule. The results are discussed in terms of the G protein antigenic structure.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The reactivities of eighteen monoclonal antibodies with different glycosylated forms of the human respiratory syncytial virus G protein were tested in Western blots, emphasizing the importance of this type of modification for the antigenicity of the mature molecule."}},"tag":"EXP"},{"id":328,"details":{"paperId":"7852ac66ac27eba639a3ba803fb6ac15ca210e16","externalIds":{"MAG":"2166677815","DOI":"10.1128/JVI.06744-11","CorpusId":"2422238","PubMed":"22318136"},"title":"The Human Respiratory Syncytial Virus Matrix Protein Is Required for Maturation of Viral Filaments","abstract":"ABSTRACT An experimental system was developed to generate infectious human respiratory syncytial virus (HRSV) lacking matrix (M) protein expression (M-null virus) from cDNA. The role of the M protein in virus assembly was then examined by infecting HEp-2 and Vero cells with the M-null virus and assessing the impact on infectious virus production and viral protein trafficking. In the absence of M, the production of infectious progeny was strongly impaired. Immunofluorescence (IF) microscopy analysis using antibodies against the nucleoprotein (N), attachment protein (G), and fusion protein (F) failed to detect the characteristic virus-induced cell surface filaments, which are believed to represent infectious virions. In addition, a large proportion of the N protein was detected in viral replication factories termed inclusion bodies (IBs). High-resolution analysis of the surface of M-null virus-infected cells by field emission scanning electron microscopy (SEM) revealed the presence of large areas with densely packed, uniformly short filaments. Although unusually short, these filaments were otherwise similar to those induced by an M-containing control virus, including the presence of the viral G and F proteins. The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures. In addition, the absence of mature viral filaments and the simultaneous increase in the level of the N protein within IBs suggest that the M protein is involved in the transport of viral ribonucleoprotein (RNP) complexes from cytoplasmic IBs to sites of budding.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The abundance of the short, stunted filaments in the absence of M indicates that M is not required for the initial stages of filament formation but plays an important role in the maturation or elongation of these structures."}},"tag":"EXP"},{"id":183,"details":{"paperId":"7760766b822cfba2e6e4f4a9a7fb3056c6c80cfb","externalIds":{"PubMedCentral":"3418853","MAG":"2134001115","DOI":"10.1074/mcp.M111.015909","CorpusId":"2925224","PubMed":"22322095"},"title":"The Human Respiratory Syncytial Virus Nonstructural Protein 1 Regulates Type I and Type II Interferon Pathways*","abstract":"Respiratory syncytial viruses encode a nonstructural protein (NS1) that interferes with type I and III interferon and other antiviral responses. Proteomic studies were conducted on human A549 type II alveolar epithelial cells and type I interferon-deficient Vero cells (African green monkey kidney cells) infected with wild-type and NS1-deficient clones of human respiratory syncytial virus to identify other potential pathway and molecular targets of NS1 interference. These analyses included two-dimensional differential gel electrophoresis and quantitative Western blotting. Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection. Because SOD2 is not directly inducible by type I interferons, it served as a marker to probe the impact of NS1 on signaling of other cytokines known to induce SOD2 expression and/or indirect effects of type I interferon signaling. Deductive analysis of results obtained from cell infection and cytokine stimulation studies indicated that interferon-γ signaling was a potential target of NS1, possibly as a result of modulation of STAT1 levels. However, this was not sufficient to explain the magnitude of the impact of NS1 on SOD2 induction in A549 cells. Vero cell infection experiments indicated that NS1 targeted a component of the type I interferon response that does not directly induce SOD2 expression but is required to induce another initiator of SOD2 expression. STAT2 was ruled out as a target of NS1 interference using quantitative Western blot analysis of infected A549 cells, but data were obtained to indicate that STAT1 was one of a number of potential targets of NS1. A label-free mass spectrometry-based quantitative approach is proposed as a means of more definitive identification of NS1 targets.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Surprisingly, NS1 was found to suppress the induction of manganese superoxide dismutase (SOD2) expression in A549 cells and to a much lesser degree Vero cells in response to infection."}},"tag":"EXP"},{"id":490,"details":{"paperId":"03232171e5d77accf4f30783ab801c2157b5a3dc","externalIds":{"PubMedCentral":"7699471","MAG":"3099516861","DOI":"10.3390/ijms21228766","CorpusId":"227168068","PubMed":"33233493"},"title":"The Importance of AGO 1 and 4 in Post-Transcriptional Gene Regulatory Function of tRF5-GluCTC, an Respiratory Syncytial Virus-Induced tRNA-Derived RNA Fragment","abstract":"Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants, the elderly, and immune-compromised patients. It is also a significant contributor to upper respiratory tract infection in the pediatric population. However, its disease mechanisms are still largely unknown. We have recently shown that a tRNA-derived RNA fragment (tRF) from the 5′-end of mature tRNA encoding GluCTC (tRF5-GluCTC), a recently discovered non-coding RNA, is functionally important for RSV replication and host gene regulation at the post-transcriptional level. However, how tRF5-GluCTC carries out the gene regulation is not fully known. In this study, we found that tRF5-GluCTC has impaired gene trans-silencing function in cells deficient of AGO1 or 4, while AGO2 and 3 seem not involved in tRF5-GluCTC-mediated gene regulation. By pulling down individual AGO protein, we discovered that tRF5-GluCTC is detectable only in the AGO4 complex, confirming the essential role of AGO4 in gene regulation and also suggesting that AGO1 contributes to the gene trans-silencing activity of tRF5-GluCTC in an atypical way. We also found that the P protein of RSV is associated with both AGO1 and 4 and AGO4 deficiency leads to reduced infectious viral particles. In summary, this study demonstrates the importance of AGO1 and 4 in mediating the gene trans-silencing function of tRF5-GluCTC.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that tRF5-GluCTC has impaired gene trans-silencing function in cells deficient of AGO1 or 4, while AGO2 and 3 seem not involved in tRF 5-GLUCTC-mediated gene regulation."}},"tag":"EXP"},{"id":146,"details":{"paperId":"74e64ca05bb0af1c1878d5ac0262f4c1b3f859c3","externalIds":{"MAG":"2981639370","PubMedCentral":"6813310","DOI":"10.1038/s41598-019-51746-0","CorpusId":"204861347","PubMed":"31649314"},"title":"The Interactome analysis of the Respiratory Syncytial Virus protein M2-1 suggests a new role in viral mRNA metabolism post-transcription","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results strongly suggest that M2-1 interacts with viral mRNA and mRNA metabolism factors from transcription to translation, and imply that M 2-1 may have an additional role in the fate of viral mRNA downstream of transcription."}},"tag":"EXP"},{"id":284,"details":{"paperId":"e73a7a65f634cea124d11f68e6ad462a0c5dddbf","externalIds":{"MAG":"2135921282","DOI":"10.1128/JVI.00460-12","CorpusId":"44856626","PubMed":"22593156"},"title":"The Interactome of the Human Respiratory Syncytial Virus NS1 Protein Highlights Multiple Effects on Host Cell Biology","abstract":"ABSTRACT Viral proteins can have multiple effects on host cell biology. Human respiratory syncytial virus (HRSV) nonstructural protein 1 (NS1) is a good example of this. During the virus life cycle, NS1 can act as an antagonist of host type I and III interferon production and signaling, inhibit apoptosis, suppress dendritic cell maturation, control protein stability, and regulate transcription of host cell mRNAs, among other functions. It is likely that NS1 performs these different roles through interactions with multiple host cell proteins. To investigate this and identify cellular proteins that could interact with NS1, we used quantitative proteomics in combination with green fluorescent protein (GFP)-trap immunoprecipitation and bioinformatic analysis. This analysis identified 221 proteins that were potentially part of complexes that could interact with NS1, with many of these associated with transcriptional regulation as part of the mediator complex, cell cycle regulation, and other functions previously assigned to NS1. Specific immunoprecipitation using the GFP trap was used to confirm the ability of selected cellular proteins to interact individually with NS1. Infection of A549 cells with recombinant viruses deficient in the expression of NS1 and overexpression analysis both demonstrated that NS1 was necessary and sufficient for the enrichment of cells in the G1 phase of the cell cycle.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Quantitative proteomics in combination with green fluorescent protein (GFP)-trap immunoprecipitation and bioinformatic analysis identified 221 proteins that were potentially part of complexes that could interact with NS1, with many of these associated with transcriptional regulation as part of the mediator complex, cell cycle regulation, and other functions previously assigned to NS1."}},"tag":"EXP"},{"id":170,"details":{"paperId":"4b9b5522b6b7b0b04ee33daf82d1e2e28246810d","externalIds":{"MAG":"1974546818","DOI":"10.1073/PNAS.96.20.11259","CorpusId":"1682526","PubMed":"10500164"},"title":"The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription.","abstract":"The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2-1) and the 90-aa M2-2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2-2 was ablated, identifying it as an accessory factor dispensable for growth in vitro. Virus lacking M2-2 grew less efficiently than did the wild-type parent in vitro, with titers that were reduced 1, 000-fold during the initial 2-5 days and 10-fold by days 7-8. Compared with wild-type virus, the intracellular accumulation of RNA by M2-2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12-15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2-2 knockout viruses. These findings suggest that M2-2 mediates a regulatory \"switch\" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2-2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that M2-2 mediates a regulatory \"switch\" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly."}},"tag":"EXP"},{"id":375,"details":{"paperId":"6a1fd5c17a24bfed8a256ce2a4b431c4f2788b2e","externalIds":{"MAG":"2154614767","DOI":"10.1128/JVI.76.21.10776-10784.2002","CorpusId":"13628719","PubMed":"12368320"},"title":"The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro","abstract":"ABSTRACT The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33Pi-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro and in vivo,osphorylation of P protein is required for efficient virus replicate in vitroand in vivo."}},"tag":"EXP"},{"id":496,"details":{"paperId":"5205734b95420b10a5f43e408d9b9a5033204b9e","externalIds":{"MAG":"2888589836","PubMedCentral":"6116276","DOI":"10.3390/v10080446","CorpusId":"52053822","PubMed":"30127286"},"title":"The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography","abstract":"Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 μm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"An optimized system for studying RSV structure is established by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET), and it is demonstrated that RSV is filamentous across several virus strains and cell lines by crye-ET, cryo -immuno EM, and thin section TEM techniques."}},"tag":"EXP"},{"id":350,"details":{"paperId":"d7b83b71d8a24e8045563c54a2324ebdd0db90aa","externalIds":{"MAG":"2140809133","DOI":"10.1128/JVI.72.2.1452-1461.1998","CorpusId":"5320927","PubMed":"9445048"},"title":"The NS1 Protein of Human Respiratory Syncytial Virus Is a Potent Inhibitor of Minigenome Transcription and RNA Replication","abstract":"ABSTRACT The NS1 protein (139 amino acids) is one of the two nonstructural proteins of human respiratory syncytial virus (RSV) and is encoded by a very abundant mRNA transcribed from the promoter-proximal RSV gene. The function of NS1 was unknown and was investigated here by using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins (N, P, L and M2-1) expressed from separate cotransfected plasmids. Coexpression of the NS1 cDNA strongly inhibited transcription and RNA replication mediated by the RSV polymerase, even when the level of expressed NS1 protein was substantially below that observed in RSV-infected cells. The effect depended on synthesis of NS1 protein rather than NS1 RNA alone. Transcription and both steps of RNA replication, namely, synthesis of the antigenome and the genome, appeared to be equally sensitive to inhibition. The efficiency of encapsidation of the plasmid-derived minigenome was not altered by coexpression of NS1, indicating that the inhibition occurs at a later step. In two different dicistronic minigenomes, transcription of each gene was equally sensitive to inhibition by NS1. This suggested that the gradient of transcriptional polarity was unaffected and that the effect of NS1 instead probably involves an early event such as polymerase entry on the genome. NS1-mediated inhibition of transcription and RNA replication was not affected by coexpression of the M2 mRNA, which has two open reading frames encoding the transcriptional elongation factor M2-1 and the putative negative regulatory factor M2-2. The potent nature of the NS1-mediated inhibition suggests that negative regulation is an authentic function of the NS1 protein, albeit not necessarily the only one.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Coexpression of the NS1 cDNA strongly inhibited transcription and RNA replication mediated by the RSV polymerase, even when the level of expressed NS1 protein was substantially below that observed in RSV-infected cells, suggesting that negative regulation is an authentic function of theNS1 protein, albeit not necessarily the only one."}},"tag":"EXP"},{"id":273,"details":{"paperId":"f57a584e25bd09f0c5a24d940aee7dfa90b0d044","externalIds":{"MAG":"2109422958","DOI":"10.1128/JVI.00181-06","CorpusId":"20824691","PubMed":"16731934"},"title":"The NS2 Protein of Human Respiratory Syncytial Virus Suppresses the Cytotoxic T-Cell Response as a Consequence of Suppressing the Type I Interferon Response","abstract":"ABSTRACT The NS1 and NS2 proteins of human respiratory syncytial virus (HRSV) have been shown to antagonize the type I interferon (IFN) response, an effect subject to host range constraints. We have now found that the HRSV NS2 protein strongly controls IFN induction in mouse cells in vitro, validating the use of the mouse model to study the consequences of these gene deletions on host immunity. We evaluated the effects of deleting the NS1 and/or NS2 gene on the induction of HRSV-specific pulmonary cytotoxic T lymphocytes (CTL) in BALB/c and 129S6 mice in response to intranasal infection with HRSV lacking the NS1 and/or NS2 gene and subsequent challenge with wild-type (wt) HRSV. In mice infected with HRSV lacking the NS2 gene (ΔNS2) or lacking the NS2 gene in combination with the NS1 gene (ΔNS1/2 HRSV), the magnitude of the pulmonary CTL response was substantially elevated compared to that of mice infected with wt HRSV or the ΔNS1 mutant, whether measured by binding of CD8+ cells to an HRSV-specific major histocompatibility complex class I tetramer, by measurement of CD8+ cells secreting gamma interferon (IFN-γ) in response to specific in vitro stimulation, or by a standard chromium release cell-killing assay. In contrast, in STAT1 knockout mice, which lack responsiveness to type I IFN, the level of IFN-γ-secreting CD8+ cells was not significantly different for HRSV lacking the NS2 gene, suggesting that the increase in CTL observed in IFN-responsive mice is type I IFN dependent. Thus, the NS2 protein of HRSV suppresses the CTL component of the adaptive immune response, and this appears to be a consequence of its suppression of type I IFN.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The NS2 protein of HRSV suppresses the CTL component of the adaptive immune response, and this appears to be a consequence of its suppression of type I IFN."}},"tag":"EXP"},{"id":437,"details":{"paperId":"1066ebb5517bbc9d444d0154b00c4ac0e3848d47","externalIds":{"MAG":"2077251885","PubMedCentral":"3769240","DOI":"10.1371/journal.pone.0074338","CorpusId":"8088408","PubMed":"24058549"},"title":"The Non-Structural NS1 Protein Unique to Respiratory Syncytial Virus: A Two-State Folding Monomer in Quasi-Equilibrium with a Stable Spherical Oligomer","abstract":"Human respiratory syncytial virus (hRSV) is a major infectious agent that cause pediatric respiratory disease worldwide. Considered one of the main virulence factors of hRSV, NS1 is known to suppress type I interferon response and signaling, thus favoring immune evasion. This, together with the fact that NS1 is unique to hRSV among paramyxoviruses, and that has no homology within databases, prompted us to investigate its conformational stability, equilibria and folding. Temperature cooperatively induces conformational changes leading to soluble spherical oligomers (NS1SOs) with amyloid-like or repetitive ß-sheet structures. The onset of the thermal transition is 45°C, and the oligomerization rate is increased by 25-fold from 40 to 46°C. Conformational stability analyzed by chemical perturbation of the NS1 monomer shows a two-state, highly reversible and cooperative unfolding, with a denaturant midpoint of 3.8 M, and a free energy change of 9.6±0.9 kcal⋅mol−1. However, two transitions were observed in the chemical perturbation of NS1SOs: the first, from 2.0 to 3.0 M of denaturant, corresponds to a conformational transition and dissociation of the oligomers to the native monomer, indicating a substantial energy barrier. The second transition (2.0 to 3.5 M denaturant) corresponds to full unfolding of the native NS1 monomer. In addition, different cosolvent perturbations converged on the formation of ß-sheet enriched soluble oligomeric species, with secondary structure resembling those obtained after mild temperature treatment. Thus, a unique protein without homologs, structure or mechanistic information may switch between monomers and oligomers in conditions compatible with the cellular environment and be potentially modulated by crowding or compartmentalization. NS1 may act as a reservoir for increased levels and impact on protein turnover.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A unique protein without homologs, structure or mechanistic information may switch between monomers and oligomers in conditions compatible with the cellular environment and be potentially modulated by crowding or compartmentalization."}},"tag":"EXP"},{"id":349,"details":{"paperId":"1877748c36cf7c02eed5a2a5b699730f78ae7582","externalIds":{"MAG":"1600427234","DOI":"10.1128/JVI.72.1.520-526.1998","CorpusId":"1508750","PubMed":"9420254"},"title":"The Product of the Respiratory Syncytial Virus M2 Gene ORF1 Enhances Readthrough of Intergenic Junctions during Viral Transcription","abstract":"ABSTRACT The mRNA encoding the M2 protein of respiratory syncytial (RS) virus contains two open reading frames (ORFs). ORF1 encodes the 22-kDa structural protein, M2, and ORF2 has the potential to encode a 10-kDa protein (90 amino acids). Using a vaccinia virus T7 expression system, we examined the RNA synthetic activities of mono- and dicistronic subgenomic replicons of RS virus by direct metabolic labeling of RNA in the presence and absence of the products of ORF1 and ORF2. In the absence of ORF1 and ORF2, the negative- and positive-sense products of genomic RNA replication and positive-sense polyadenylated mRNA(s) were synthesized. Expression of the whole M2 transcription unit (containing ORF1 and ORF2) or ORF1 alone caused an increase in the synthesis of polyadenylated mRNA, the majority of which was due to a substantial increase in the quantity of polycistronic mRNAs generated by the polymerase failing to terminate at gene end signals. In agreement with previous reports, the ORF2 product was found to inhibit viral RNA replication and mRNA transcription. These data show that the M2 protein functions as a transcriptional antiterminator that enhances the ability of the viral RNA polymerase to read through intergenic junctions. The role of such a function during the viral life cycle is discussed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data show that the M2 protein functions as a transcriptional antiterminator that enhances the ability of the viral RNA polymerase to read through intergenic junctions and inhibit viral RNA replication and mRNA transcription."}},"tag":"EXP"},{"id":55,"details":{"paperId":"abf232dafdf3bd1b94a3cd8aa8d261d00cd2e9e9","externalIds":{"MAG":"2031702365","DOI":"10.1016/J.BBRC.2007.11.042","CorpusId":"41721136","PubMed":"18036342"},"title":"The RSV F and G glycoproteins interact to form a complex on the surface of infected cells.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Data suggest the existence of multiple virus glycoprotein complexes within the RSV envelope, and the association between the G and F proteins suggest an indirect role for the G protein in this process."}},"tag":"EXP"},{"id":70,"details":{"paperId":"bc51a6cc024ea84347ea67f87bb4dfaa8fe02c15","externalIds":{"MAG":"1981618408","DOI":"10.1016/j.micinf.2012.07.015","CorpusId":"21610820","PubMed":"22884716"},"title":"The RSV fusion receptor: not what everyone expected it to be.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Current knowledge about respiratory syncytial virus binding and entry into cells is reviewed, and the recent discovery of Nucleolin as a fusion receptor for RSV opens new avenues for developing interventions."}},"tag":"EXP"},{"id":321,"details":{"paperId":"e5c140e0a212fcf4740c3f3d2649042957d8841f","externalIds":{"MAG":"2037203429","DOI":"10.1128/JVI.03086-12","CorpusId":"3230457","PubMed":"23903836"},"title":"The Respiratory Syncytial Virus Fusion Protein Targets to the Perimeter of Inclusion Bodies and Facilitates Filament Formation by a Cytoplasmic Tail-Dependent Mechanism","abstract":"ABSTRACT The human respiratory syncytial virus (HRSV) fusion (F) protein cytoplasmic tail (CT) and matrix (M) protein are key mediators of viral assembly, but the underlying mechanisms are poorly understood. A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production. The ability of F mutants with alanine substitutions in the CT to complement an F-null virus in generating infectious progeny was quantitated by flow cytometry. Two CT regions with impact on infectious progeny production were identified: residues 557 to 566 (CT-R1) and 569 to 572 (CT-R2). Substitutions in CT-R1 decreased infectivity by 40 to 85% and increased the level of F-induced cell-cell fusion but had little impact on assembly of viral surface filaments, which are believed to be virions. Substitutions in CT-R2, as well as deletion of the entire CT, abrogated infectious progeny production and impaired viral filament formation. However, CT-R2 mutations did not block but rather delayed the formation of viral filaments, which continued to form at a low rate and contained the viral M protein and nucleoprotein (N). Microscopy analysis revealed that substitutions in CT-R2 but not CT-R1 led to accumulation of M and F proteins within and at the perimeter of viral inclusion bodies (IBs), respectively. The accumulation of M and F at IBs and coincident strong decrease in filament formation and infectivity upon CT-R2 mutations suggest that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A complementation assay was developed to systematically examine the role of the F protein CT in infectious virus production and suggested that F interaction with IBs is an important step in the virion assembly process and that CT residues 569 to 572 act to facilitate release of M-ribonucleoprotein complexes from IBs."}},"tag":"EXP"},{"id":298,"details":{"paperId":"5934674f07df36a91356f2b260a2c841611d7391","externalIds":{"MAG":"1979400047","DOI":"10.1128/JVI.01347-13","CorpusId":"37712763","PubMed":"23843644"},"title":"The Respiratory Syncytial Virus Fusion Protein and Neutrophils Mediate the Airway Mucin Response to Pathogenic Respiratory Syncytial Virus Infection","abstract":"ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of death due to a viral etiology in infants. RSV disease is characterized by epithelial desquamation, neutrophilic bronchiolitis and pneumonia, and obstructive pulmonary mucus. It has been shown that infection of BALB/cJ mice with RSV clinical isolate A2001/2-20 (2-20) results in a higher early viral load, greater airway necrosis, and higher levels of interleukin-13 (IL-13) and airway mucin expression than infection with RSV laboratory strain A2. We hypothesized that the fusion (F) protein of RSV 2-20 is a mucus-inducing viral factor. In vitro, the fusion activity of 2-20 F but not that of A2 F was enhanced by expression of RSV G. We generated a recombinant F-chimeric RSV by replacing the F gene of A2 with the F gene of 2-20, generating A2–2-20F. Similar to the results obtained with the parent 2-20 strain, infection of BALB/cJ mice with A2–2-20F resulted in a higher early viral load and higher levels of subsequent pulmonary mucin expression than infection with the A2 strain. A2–2-20F infection induced greater necrotic airway damage and neutrophil infiltration than A2 infection. We hypothesized that the neutrophil response to A2–2-20F infection is involved in mucin expression. Antibody-mediated depletion of neutrophils in RSV-infected mice resulted in lower tumor necrosis factor alpha levels, fewer IL-13-expressing CD4 T cells, and less airway mucin production in the lung. Our data are consistent with a model in which the F and attachment (G) glycoprotein functional interaction leads to enhanced fusion and F is a key factor in airway epithelium infection, pathogenesis, and subsequent airway mucin expression.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"A2–2-20F infection induced greater necrotic airway damage and neutrophil infiltration than A2 infection, consistent with a model in which the F and attachment glycoprotein functional interaction leads to enhanced fusion and F is a key factor in airway epithelium infection, pathogenesis, and subsequent airway mucin expression."}},"tag":"EXP"},{"id":277,"details":{"paperId":"1425b8b93f1fb3ad5c21f5496d0b1589eeb27843","externalIds":{"MAG":"2009631060","DOI":"10.1128/JVI.00335-09","CorpusId":"43439973","PubMed":"19386701"},"title":"The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner","abstract":"ABSTRACT The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely α-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and ∼7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative α-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1."}},"tag":"EXP"},{"id":256,"details":{"paperId":"c7da47a124380b11a67c747ac94b9970f0274a23","externalIds":{"DOI":"10.1101/2023.01.02.522538","CorpusId":"255441900"},"title":"The Respiratory Syncytial Virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly","abstract":"The M2-2 protein from the respiratory syncytial virus (RSV) is a 10 kDa protein expressed by the second ORF of the viral gene M2. During infection, M2-2 has been described as the polymerase cofactor responsible for promoting genome replication. This function was first inferred by infection with a mutant virus lacking the M2-2 ORF, in which viral genome presented delayed accumulation in comparison to wild-type virus. In accordance with this phenotype, it has been recently shown that M2-2 promotes changes in interactions between the polymerase and other viral proteins at early stages of infection. Despite its well-explored role in the regulation of the polymerase activity, little has been made to investigate the relationship of M2-2 with cellular proteins. In fact, a previous report showed poor recruitment of M2-2 to viral structures, with the protein being mainly localized to the nucleus and cytoplasmic granules. To unravel which other functions M2-2 exerts during infection, we expressed the protein in HEK293T cells and performed proteomic analysis of co-immunoprecipitated partners, identifying enrichment of proteins involved with regulation of translation, protein folding and mRNA splicing. In approaches based on these data, we found that M2-2 expression downregulates eiF2α phosphorylation and inhibits stress granules assembly under arsenite induction. In addition, we also verified that M2-2 inhibits translation initiation, and is targeted for proteasome degradation, being localized to granules composed by defective ribosomal products at the cytoplasm. These results suggest that besides its functions in the regulation of genome replication, M2-2 may exert additional functions to contribute to successful RSV infection. Author summary Exploring how viruses take control of the cellular machinery is a common strategy to understand the infection process and to identify targets for inhibition of virus replication. In this work we investigated the cellular functions of the protein M2-2 from the respiratory syncytial virus. Although this virus is an important pathogen responsible for respiratory infections in immunocompromised individuals, currently there are no vaccines or effective treatments to inhibit its infection. Our findings showed that the protein M2-2 interferes with protein synthesis, being able to downregulate the assembly of stress granules during stress stimuli. Besides, we verified the relationship between M2-2 and the proteasome machinery, which is responsible for protein degradation and is also involved with protein synthesis. These results present new functions for the protein M2-2, indicating additional mechanisms utilized by the virus to facilitate infection, providing new perspectives for the search of antiviral targets.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is shown that the protein M2-2 interferes with protein synthesis, being able to downregulate the assembly of stress granules during stress stimuli, and additional mechanisms utilized by the virus to facilitate infection are indicated."}},"tag":"EXP"},{"id":315,"details":{"paperId":"2934a0bda45a43394518380f5e6eadcfec4c6e85","externalIds":{"MAG":"2008787934","DOI":"10.1128/JVI.02374-08","CorpusId":"23208932","PubMed":"19297465"},"title":"The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism","abstract":"ABSTRACT The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M's ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for relatedParamyxoviruses."}},"tag":"EXP"},{"id":294,"details":{"paperId":"1cad575e31ee2c7369a6fe7a77e74d52b43c4477","externalIds":{"MAG":"2522992607","DOI":"10.1128/JVI.01193-16","CorpusId":"19407204","PubMed":"27654298"},"title":"The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation","abstract":"ABSTRACT Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology."}},"tag":"EXP"},{"id":446,"details":{"paperId":"2e6ecde29277cd38adf9fa954d7b504bca008aa9","externalIds":{"MAG":"2085811591","PubMedCentral":"3475672","DOI":"10.1371/journal.ppat.1002980","CorpusId":"7809935","PubMed":"23093940"},"title":"The Respiratory Syncytial Virus Polymerase Has Multiple RNA Synthesis Activities at the Promoter","abstract":"Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection."}},"tag":"EXP"},{"id":304,"details":{"paperId":"1ff21966ef52fd516a903cbd3b1f0fa9d45698cf","externalIds":{"MAG":"1991100819","DOI":"10.1128/JVI.01604-08","CorpusId":"2044617","PubMed":"18842713"},"title":"The Secreted Form of Respiratory Syncytial Virus G Glycoprotein Helps the Virus Evade Antibody-Mediated Restriction of Replication by Acting as an Antigen Decoy and through Effects on Fc Receptor-Bearing Leukocytes","abstract":"ABSTRACT Respiratory syncytial virus (RSV) readily infects and reinfects during infancy and throughout life, despite maternal antibodies and immunity from prior infection and without the need for significant antigenic change. RSV has two neutralization antigens, the F and G virion glycoproteins. G is expressed in both membrane-bound (mG) and secreted (sG) forms. We investigated whether sG might act as a decoy for neutralizing antibodies by comparing the in vitro neutralization of wild-type (wt) RSV versus recombinant mG RSV expressing only mG. wt RSV indeed was less susceptible than mG RSV to monovalent G-specific and polyvalent RSV-specific antibodies, whereas susceptibility to F-specific antibodies was equivalent. This difference disappeared when the virus preparations were purified to remove sG. Thus, sG appears to function as a neutralization decoy. We evaluated this effect in vivo in mice by comparing the effects of passively transferred antibodies on the pulmonary replication of wt RSV versus mG RSV. Again, wt RSV was less sensitive than mG RSV to G-specific and RSV-specific antibodies; however, a similar difference was also observed with F-specific antibodies. This confirmed that sG helps wt RSV evade the antibody-dependent restriction of replication but indicated that in mice, it is not acting primarily as a decoy for G-specific antibodies, perhaps because sG is produced in insufficient quantities in this poorly permissive animal. Rather, we found that the greater sensitivity of mG versus wt RSV to the antiviral effect of passively transferred RSV antibodies required the presence of inflammatory cells in the lung and was Fcγ receptor dependent. Thus, sG helps RSV escape the antibody-dependent restriction of replication via effects as an antigen decoy and as a modulator of leukocytes bearing Fcγ receptors.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is confirmed that sG helps wt RSV evade the antibody-dependent restriction of replication via effects as an antigen decoy and as a modulator of leukocytes bearing Fcγ receptors."}},"tag":"EXP"},{"id":293,"details":{"paperId":"f0f1fee5ab4cae04ba2da747db1fb399d9dbc5c8","externalIds":{"MAG":"1983091270","DOI":"10.1128/JVI.01162-12","CorpusId":"32990858","PubMed":"22837211"},"title":"The Secreted G Protein of Human Respiratory Syncytial Virus Antagonizes Antibody-Mediated Restriction of Replication Involving Macrophages and Complement","abstract":"ABSTRACT The respiratory syncytial virus (RSV) G and F glycoproteins are the neutralization antigens, and G also is expressed in a soluble form (sG). Previously, sG was demonstrated to reduce the efficiency of RSV antibody-mediated neutralization by serving as an antigen decoy and to inhibit the antibody-mediated antiviral effects of Fc receptor-bearing leukocytes. The present study demonstrated that effective antibody-mediated restriction in vivo, and the evasion of this restriction by sG, involves pulmonary macrophages and complement, but not neutrophils.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that effective antibody-mediated restriction in vivo, and the evasion of this restriction by sG, involves pulmonary macrophages and complement, but not neutrophils."}},"tag":"EXP"},{"id":174,"details":{"paperId":"5a07810060221b6f951b92052aded5389255214a","externalIds":{"MAG":"2074242518","DOI":"10.1074/jbc.M111.332791","CorpusId":"23762998","PubMed":"22621926"},"title":"The Small Hydrophobic Protein of the Human Respiratory Syncytial Virus Forms Pentameric Ion Channels*","abstract":"Background: Few effective treatments exist for human respiratory syncytial virus infection. The absence of small hydrophobic (SH) protein in RSV leads to viral attenuation. Results: SH protein forms pentamers and shows pH-dependent ion channel activity. Conclusion: SH protein forms pentameric ion channels. Significance: The SH protein and its channel activity constitute a potential drug target. The small hydrophobic (SH) protein is encoded by the human respiratory syncytial virus. Its absence leads to viral attenuation in the context of whole organisms, and it prevents apoptosis in infected cells. Herein, we have examined the structure of SH protein in detergent micelles and in lipid bilayers, by solution NMR and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. We found that SH protein has a single α-helical transmembrane domain and forms homopentamers in several detergents. In detergent micelles, the transmembrane domain is flanked N-terminally by an α-helix that forms a ring around the lumen of the pore and C-terminally by an extended β-turn. SH protein was found in the plasma membrane of transiently expressing HEK 293 cells, which showed pH-dependent (acid-activated) channel activity. Channel activity was abolished in mutants lacking both native His residues, His22 and His51, but not when either His was present. Herein, we propose that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication. Viroporins are short (∼100 amino acids) viral membrane proteins that form oligomers of a defined size, act as proton or ion channels, and in general enhance membrane permeability in the host. However, with some exceptions, their precise biological role of their channel activity is not understood. In general, viroporins resemble poorly specialized proteins but are nevertheless critical for viral fitness. In vivo, viruses lacking viroporins usually exhibit an attenuated or weakened phenotype, altered tropism, and diminished pathological effects. We have chosen to study the SH protein, 64 amino acids long, found in the human respiratory syncytial virus because of the effect of RSV on human health and the lack of adequate antivirals. We show that SH protein forms oligomers that behave as ion channels when activated at low pH. This study adds SH protein to a growing group of viroporins that have been structurally characterized. Although the precise biological role of this pentameric channel is still unknown, this report is nevertheless essential to fill some of the many gaps that exist in the understanding of SH protein function.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is proposed that the pentameric model of SH protein presented is a physiologically relevant conformation, albeit probably not the only one, in which SH contributes to RSV infection and replication."}},"tag":"EXP"},{"id":387,"details":{"paperId":"83ca606131d9d28bd72824e474d4ddbcb53d7189","externalIds":{"MAG":"1992111405","DOI":"10.1128/JVI.78.7.3524-3532.2004","CorpusId":"25234819","PubMed":"15016875"},"title":"The Soluble Form of Human Respiratory Syncytial Virus Attachment Protein Differs from the Membrane-Bound Form in Its Oligomeric State but Is Still Capable of Binding to Cell Surface Proteoglycans","abstract":"ABSTRACT The soluble (Gs) and membrane-bound (Gm) forms of human respiratory syncytial virus (HRSV) attachment protein were purified by immunoaffinity chromatography from cultures of HEp-2 cells infected with vaccinia virus recombinants expressing either protein. Sucrose gradient centrifugation indicated that Gs, which is secreted into the culture medium, remains monomeric, whereas Gm is an oligomer, probably a homotetramer. Nevertheless, Gs was capable of binding to the surface of cells in vitro, as assessed by a flow cytometry-based binding assay. The attachment of Gs to cells was inhibited by previous heparinase treatment of living cells, and Gs did not bind to CHO cell mutants defective in proteoglycan biosynthesis. Thus, Gs, as previously reported for the G protein of intact virions, binds to glycosaminoglycans presented at the cell surface as proteoglycans. Deletion of a previously reported heparin binding domain from Gs protein substantially inhibited its ability to bind to cells, but the remaining level of binding was still sensitive to heparinase treatment, suggesting that other regions of the Gs molecule may contribute to attachment to proteoglycans. The significance of these results for HRSV infection is discussed.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Gs, as previously reported for the G protein of intact virions, binds to glycosaminoglycans presented at the cell surface as proteoglyCans, and the significance of these results for HRSV infection is discussed."}},"tag":"EXP"},{"id":398,"details":{"paperId":"1b3bd490958845a5b2a9e4d99383bbfffa910cd2","externalIds":{"PubMedCentral":"6234862","MAG":"2901596222","DOI":"10.1128/mBio.01554-18","CorpusId":"53306005","PubMed":"30425144"},"title":"The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex","abstract":"Human respiratory syncytial virus (HRSV) is a leading cause of respiratory illness, particularly in the young, elderly, and immunocompromised, and has also been linked to the development of asthma. HRSV replication depends on P and L, whereas transcription also requires M2-1. M2-1 interacts with P and RNA at overlapping binding sites; while these interactions are necessary for transcriptional activity, the mechanism of M2-1 action is unclear. To better understand HRSV transcription, we solved the crystal structure of M2-1 in complex with the minimal P interaction domain, revealing molecular details of the M2-1/P interface and defining the orientation of M2-1 within the tripartite complex. The M2-1/P interaction is relatively weak, suggesting high-affinity RNAs may displace M2-1 from the complex, providing the basis for a new model describing the role of M2-1 in transcription. Recently, the small molecules quercetin and cyclopamine have been used to validate M2-1 as a drug target. ABSTRACT Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause life-threatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90–110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences. IMPORTANCE Human respiratory syncytial virus (HRSV) is a leading cause of respiratory illness, particularly in the young, elderly, and immunocompromised, and has also been linked to the development of asthma. HRSV replication depends on P and L, whereas transcription also requires M2-1. M2-1 interacts with P and RNA at overlapping binding sites; while these interactions are necessary for transcriptional activity, the mechanism of M2-1 action is unclear. To better understand HRSV transcription, we solved the crystal structure of M2-1 in complex with the minimal P interaction domain, revealing molecular details of the M2-1/P interface and defining the orientation of M2-1 within the tripartite complex. The M2-1/P interaction is relatively weak, suggesting high-affinity RNAs may displace M2-1 from the complex, providing the basis for a new model describing the role of M2-1 in transcription. Recently, the small molecules quercetin and cyclopamine have been used to validate M2-1 as a drug target.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"To better understand HRSV transcription, the crystal structure of M2-1 in complex with the minimal P interaction domain is solved, revealing molecular details of the M 2-1/P interface and defining the orientation of M1-1 within the tripartite complex."}},"tag":"EXP"},{"id":325,"details":{"paperId":"a68767c4af9613284aa5eccfcc5366169ebc928e","externalIds":{"MAG":"2001655298","DOI":"10.1128/JVI.03856-13","CorpusId":"206817622","PubMed":"24672034"},"title":"The Thr205 Phosphorylation Site within Respiratory Syncytial Virus Matrix (M) Protein Modulates M Oligomerization and Virus Production","abstract":"ABSTRACT Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. IMPORTANCE We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments."}},"tag":"EXP"},{"id":391,"details":{"paperId":"0ee4efb1d4235447b20fd1d245dc8159ceb3721b","externalIds":{"MAG":"2101954261","DOI":"10.1128/JVI.79.19.12528-12535.2005","CorpusId":"18107313","PubMed":"16160180"},"title":"The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence","abstract":"ABSTRACT The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells."}},"tag":"EXP"},{"id":25,"details":{"paperId":"9828651735013c24013984334493ac7aa1dc0fb8","externalIds":{"MAG":"162404543","DOI":"10.1007/978-1-4615-3790-8_14","CorpusId":"82763720"},"title":"The Unusual Attachment Glycoprotein of the Respiratory Syncytial Viruses","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The purpose of this chapter is to describe the structure and function of the G protein and to discuss its role in infection and immunity to RS virus disease."}},"tag":"EXP"},{"id":457,"details":{"paperId":"4867e0d05f067189d610ca8e764d52dcc50a7b49","externalIds":{"MAG":"2966329493","PubMedCentral":"6695199","DOI":"10.1371/journal.ppat.1007963","CorpusId":"199449168","PubMed":"31381610"},"title":"The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells","abstract":"Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is identified that ATP1A1 is a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and PST2238 is identified as a potential anti-RSV drug."}},"tag":"EXP"},{"id":226,"details":{"paperId":"68a4d724d5c7bb090f863044aaeb3edf46d2c39b","externalIds":{"MAG":"1769966102","DOI":"10.1099/0022-1317-81-1-129","CorpusId":"20296492","PubMed":"10640550"},"title":"The bulk of the phosphorylation of human respiratory syncytial virus phosphoprotein is not essential but modulates viral RNA transcription and replication.","abstract":"The ability of variants of the human respiratory syncytial virus (HRSV) phosphoprotein (P protein) to support RNA transcription and replication has been studied by using HRSV-based subgenomic replicons. The serine residues normally phosphorylated in P during HRSV infection have been replaced by other residues. The results indicate that the bulk of phosphorylation of P (98%) is not essential for viral RNA transcription or replication but that phosphorylation can modulate these processes.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results indicate that the bulk of phosphorylation of P (98%) is not essential for viral RNA transcription or replication but thatosphorylation can modulate these processes."}},"tag":"EXP"},{"id":154,"details":{"paperId":"d8f472a16b6a37aa908fb5bcedbfba12780b23dc","externalIds":{"MAG":"2097194611","DOI":"10.1073/PNAS.0409478102","CorpusId":"7208623","PubMed":"15956195"},"title":"The cysteine-rich region of respiratory syncytial virus attachment protein inhibits innate immunity elicited by the virus and endotoxin.","abstract":"The attachment protein (glycoprotein) of respiratory syncytial virus (RSV) has long been associated with disease potentiation and respiratory symptoms. The glycoprotein has a conserved cysteine-rich region (GCRR) whose function is unknown and which is not necessary for efficient viral replication. In this report, we show that the GCRR is a powerful inhibitor of the innate immune response against RSV, and that early secretion of glycoprotein is critical to modulate inflammation after RSV infection. Importantly, the GCRR is also a potent inhibitor of cytokine production mediated by several TLR agonists, indicating that this peptide sequence displays broad antiinflammatory properties. These findings have important implications for RSV pathogenesis and describe an inhibitor of TLR-mediated inflammatory responses that could have clinical applications.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is shown that the GCRR is a powerful inhibitor of the innate immune response against RSV, and that early secretion of glycoprotein is critical to modulate inflammation after RSV infection."}},"tag":"EXP"},{"id":246,"details":{"paperId":"dc3b8f8eb56c27519804c0dc41a0625b65142817","externalIds":{"MAG":"1985600554","DOI":"10.1099/VIR.0.81481-0","CorpusId":"11194119","PubMed":"16432027"},"title":"The cytoplasmic domain of the F protein of Human respiratory syncytial virus is not required for cell fusion.","abstract":"The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion."}},"tag":"EXP"},{"id":427,"details":{"paperId":"b181047b067b904355f348752e1c56a03b2b4cb9","externalIds":{"DOI":"10.1186/s12985-018-1091-7","CorpusId":"255963036"},"title":"The eukaryotic translation elongation factor 1A regulation of actin stress fibers is important for infectious RSV production","abstract":null,"publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"The cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release."}},"tag":"EXP"},{"id":432,"details":{"paperId":"0eee7cf3439d6a83d8faea90cfb189e392d06dbd","externalIds":{"MAG":"2153381915","DOI":"10.1261/rna.2813411","CorpusId":"19116134","PubMed":"21878549"},"title":"The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus.","abstract":"There is limited knowledge regarding how the RNA-dependent RNA polymerases of the nonsegmented negative-strand RNA viruses initiate genome replication. In a previous study of respiratory syncytial virus (RSV) RNA replication, we found evidence that the polymerase could select the 5'-ATP residue of the genome RNA independently of the 3' nucleotide of the template. To investigate if a similar mechanism is used during antigenome synthesis, a study of initiation from the RSV leader (Le) promoter was performed using an intracellular minigenome assay in which RNA replication was restricted to a single step, so that the products examined were derived only from input mutant templates. Templates in which Le nucleotides 1U, or 1U and 2G, were deleted directed efficient replication, and in both cases, the replication products were initiated at the wild-type position, at position -1 or -2 relative to the template, respectively. Sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence. These data indicate that the RSV polymerase is able to select the first two nucleotides of the antigenome and initiate at the correct position, even if the 3'-terminal two nucleotides of the template are missing. Substitution of positions +1 and +2 of the template reduced RNA replication and resulted in increased initiation at positions +3 and +5. Together these data suggest a model for how the RSV polymerase initiates antigenome synthesis.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Analysis of initiation from the RSV leader (Le) promoter and sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence and suggest a model for how theRSV polymerase initiates antigenome synthesis."}},"tag":"EXP"},{"id":75,"details":{"paperId":"2a95b301721f8cda3d2456d1ab162003226fbc7a","externalIds":{"MAG":"2525755163","DOI":"10.1016/j.vaccine.2016.09.030","CorpusId":"4628231","PubMed":"27686836"},"title":"The innate immune response to RSV: Advances in our understanding of critical viral and host factors.","abstract":null,"publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Newly discovered host pathways involved in the innate response to RSV, together with the potential generation of DVG-derived oligonucleotides, present various novel opportunities for the design of vaccine adjuvants able to induce a protective response against RSV and similar viruses."}},"tag":"EXP"},{"id":461,"details":{"paperId":"a1443416e119ee54754b0491cf9a7c872db62eee","externalIds":{"PubMedCentral":"8057581","DOI":"10.1371/journal.ppat.1009469","CorpusId":"233193790","PubMed":"33831114"},"title":"The larger attachment glycoprotein of respiratory syncytial virus produced in primary human bronchial epithelial cultures reduces infectivity for cell lines","abstract":"Respiratory syncytial virus (RSV) infects the upper and lower respiratory tracts and can cause lower respiratory tract infections in children and elders. RSV has traditionally been isolated, grown, studied and quantified in immortalized cell lines, most frequently HEp-2 cells. However, in vivo RSV infection is modeled more accurately in primary well differentiated human bronchial epithelial (HBE) cultures where RSV targets the ciliated cells and where the putative RSV receptor differs from the receptor on HEp-2 cells. The RSV attachment (G) glycoprotein in virions produced by HEp-2 cells is a highly glycosylated 95 kDa protein with a 32 kDa peptide core. However, virions produced in HBE cultures, RSV (HBE), contain an even larger, 170 kDa, G protein (LgG). Here we show that LgG is found in virions from both subgroups A and B lab-adapted and clinical isolates. Unexpectedly, RSV (HBE) virions were approximately 100-fold more infectious for HBE cultures than for HEp-2 cells. Surprisingly, the cause of this differential infectivity, was reduced infectivity of RSV (HBE) on HEp-2 cells rather than enhanced infectivity on HBE cultures. The lower infectivity of RSV(HBE) for HEp-2 cells is caused by the reduced ability of LgG to interact with heparan sulfate proteoglycans (HSPG), the RSV receptor on HEp-2 cells. The discovery of different infectivity corresponding with the larger form of the RSV attachment protein when produced by HBE cultures highlights the importance of studying a virus produced by its native host cell and the potential impact on quantifying virus infectivity on cell lines where the virus entry mechanisms differ from their natural target cell.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The discovery of different infectivity corresponding with the larger form of the RSV attachment protein when produced by HBE cultures highlights the importance of studying a virus produced by its native host cell and the potential impact on quantifying virus infectivity on cell lines where the virus entry mechanisms differ from their natural target cell."}},"tag":"EXP"},{"id":32,"details":{"paperId":"b4dbb0aa1e6f8b3c97c52f264687ea835a4cf057","externalIds":{"MAG":"1522237878","DOI":"10.1007/s00705-003-0112-y","CorpusId":"24930775","PubMed":"12827470"},"title":"The matrix protein of Human respiratory syncytial virus localises to the nucleus of infected cells and inhibits transcription","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The kinetics of localisation of matrix (M) protein of Human respiratory syncytial virus in infected cells suggests that M protein may play a role early in the infection by inhibiting host cell transcription."}},"tag":"EXP"},{"id":339,"details":{"paperId":"6dc1433095971cadee77fe5720fbbaf6c76b8032","externalIds":{"MAG":"1855993737","DOI":"10.1128/jvi.68.7.4538-4546.1994","CorpusId":"22445075","PubMed":"8207828"},"title":"The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons","abstract":"Respiratory syncytial (RS) virus synthesizes two mature forms of its attachment glycoprotein G: an anchored type II integral membrane form and a smaller form that is secreted into the medium. Here we demonstrate that these two forms are synthesized as distinct primary translation products of a single species of G protein mRNA by initiation at either of two different AUGs. Mutant cDNAs which eliminated one of the other of the two AUG codons near the 5' end of the G gene open reading frame were constructed. Analysis of the proteins synthesized from these cDNAs, either by translation of transcripts in a cell-free system or in cells infected with recombinant vaccinia viruses containing either one of the mutant cDNAs, showed that elimination of either the first or the second of these AUG codons abrogated the synthesis of the membrane-anchored or the secreted form of the protein, respectively. Additionally, two unglycosylated forms of G protein which comigrated with the unglycosylated G proteins expressed by these recombinant viruses were detected in RS virus-infected cells. Since the second AUG encodes a methionine residue that lies near the middle of the signal/anchor domain, initiation at this codon resulted in a protein with a hydrophobic amino terminus. This form of the glycoprotein was efficiently secreted from cells infected with the vaccinia virus recombinant, and the amino-terminal sequence of this protein was identical to that of G protein secreted from RS virus-infected cells. Our results demonstrate that the secreted form of RS virus G protein is produced by initiation at the second AUG codon of the G open reading frame, followed by proteolytic removal of the signal/anchor domain.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results demonstrate that the secreted form of RS virus G protein is produced by initiation at the second AUG codon of the G open reading frame, followed by proteolytic removal of the signal/anchor domain."}},"tag":"EXP"},{"id":462,"details":{"paperId":"bd3d1cf175da97ed90700d28aa5065abaa7a7bd9","externalIds":{"PubMedCentral":"8130918","DOI":"10.1371/journal.ppat.1009562","CorpusId":"233984852","PubMed":"33956914"},"title":"The methyltransferase domain of the Respiratory Syncytial Virus L protein catalyzes cap N7 and 2’-O-methylation","abstract":"Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2’-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5’ end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at μM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion."}},"tag":"EXP"},{"id":250,"details":{"paperId":"6c7f97674df666d6c7a92d2d4aa70bffa9e1b9d4","externalIds":{"MAG":"2055357560","DOI":"10.1099/VIR.0.82282-0","CorpusId":"35888801","PubMed":"17170452"},"title":"The nine C-terminal amino acids of the respiratory syncytial virus protein P are necessary and sufficient for binding to ribonucleoprotein complexes in which six ribonucleotides are contacted per N protein protomer.","abstract":"The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100-160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161-241) fused to GST pulled down exclusively RNA-N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22,900 and 24,820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5' and 3' ends.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Pull-down assays revealed that the region comprising the last nine C-terminal amino acid residues of P is sufficient for efficient binding to N and suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5' and 3' ends."}},"tag":"EXP"},{"id":91,"details":{"paperId":"18edb11de5ae77bbfb7fef3a0d158752e40e29a5","externalIds":{"MAG":"2010291418","DOI":"10.1016/j.virol.2013.11.014","CorpusId":"43876058","PubMed":"24418538"},"title":"The respiratory syncytial virus (RSV) nonstructural proteins mediate RSV suppression of glucocorticoid receptor transactivation.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that the RSV nonstructural proteins mediate RSV repression of GR-induced transactivation and that inhibition of the nonStructural proteins may be a viable target for therapy against RSV-related disease."}},"tag":"EXP"},{"id":237,"details":{"paperId":"6ab072bfaefc100b5c2e27da5254fa0285edd594","externalIds":{"PubMedCentral":"3749527","MAG":"2135413421","DOI":"10.1099/vir.0.053025-0","CorpusId":"18981027","PubMed":"23677789"},"title":"The respiratory syncytial virus nucleoprotein–RNA complex forms a left-handed helical nucleocapsid","abstract":"Respiratory syncytial virus (RSV) is an important human pathogen. Its nucleocapsid (NC), which comprises the negative sense RNA viral genome coated by the viral nucleoprotein N, is a critical assembly that serves as template for both mRNA synthesis and genome replication. We have previously described the X-ray structure of an NC-like structure: a decameric ring formed of N-RNA that mimics one turn of the helical NC. In the absence of experimental data we had hypothesized that the NC helix would be right-handed, as the N–N contacts in the ring appeared to more easily adapt to that conformation. We now unambiguously show that the RSV NC is a left-handed helix. We further show that the contacts in the ring can be distorted to maintain key N–N-protein interactions in a left-handed helix, and discuss the implications of the resulting atomic model of the helical NC for viral replication and transcription.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is unambiguously show that the RSV NC is a left-handed helix, and it is shown that the contacts in the ring can be distorted to maintain key N–N-protein interactions in aleft- handed helix."}},"tag":"EXP"},{"id":96,"details":{"paperId":"0a99a9deec2b3aba1436b747449b41c8111d0032","externalIds":{"MAG":"2988904572","DOI":"10.1016/j.virol.2019.11.002","CorpusId":"208168322","PubMed":"31739186"},"title":"The respiratory syncytial virus polymerase can perform RNA synthesis with modified primers and nucleotide analogs.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The RSV polymerase was found to accept primers containing 5' biotin or digoxygenin modifications, and nucleotide analogs that are reactive or fluorescent, including 5-ethynyl UTP, 8-azido ATP, 2-amino PTP, and thieno-GTP."}},"tag":"EXP"},{"id":242,"details":{"paperId":"6c51c1f9ae705b484252a249512bd71a63b2ac01","externalIds":{"MAG":"2121107058","DOI":"10.1099/VIR.0.80563-0","CorpusId":"15795070","PubMed":"15659757"},"title":"The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection.","abstract":"The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein was isolated from [35S]methionine- and [33P]orthophosphate-labelled RSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role of tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580, an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependent tyrosine kinase activity and that this modification influences its cellular distribution.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependent tyrosine kinase activity and that this modification influences its cellular distribution."}},"tag":"EXP"},{"id":126,"details":{"paperId":"30bc96156e05c6f76fe5985758215083ab19e904","externalIds":{"MAG":"2025615814","DOI":"10.1021/bi200661k","CorpusId":"37861089","PubMed":"21877705"},"title":"The respiratory syncytial virus transcription antiterminator M(2-1) is a highly stable, zinc binding tetramer with strong pH-dependent dissociation and a monomeric unfolding intermediate.","abstract":"The human respiratory syncytial virus M(2-1) transcription antiterminator is an essential elongation factor required by the RNA polymerase for effective transcription beyond the first two nonstructural genes. Its exclusive presence in pneumovirus among all paramyxovirus suggests a unique function within this small genus. With the aim of understanding its biochemical properties, we investigated this α-helical tetramer by making use of a biophysical approach. We found that the tetramer hydrodynamic radius is considerably extended at high ionic strengths and determined its zinc content to be one atom per monomer. Dissociation-unfolding experiments show a fully reversible and concentration-dependent cooperative transition, but secondary and tertiary structural changes are uncoupled at lower protein concentrations. We detect the presence of a monomeric intermediate, which can be classified as a \"late molten globule\" with substantial secondary and tertiary structure. Global fittings of experiments from three different probes at two M(2-1) concentrations provide a free energy of dissociation-unfolding of -36.8 ± 0.1 kcal mol(-1), corresponding to a tight dissociation constant of 10(-28) M(3) at pH 7.0. The tetramer affinity is strongly governed by pH, with a free energy change of 13 kcal mol(-1) when pH decreases from 7.0 to 5.0 (K(D) = 10(-18) M(3)). The drastic changes that take place within a pH range compatible with a cellular environment strongly suggest a regulatory effect of pH on M(2-1) structure and biochemical properties, likely affecting transcription and interaction with proteins and RNA.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is found that the tetramer hydrodynamic radius is considerably extended at high ionic strengths and its zinc content to be one atom per monomer and the presence of a monomeric intermediate, which can be classified as a \"late molten globule\" with substantial secondary and tertiary structure."}},"tag":"EXP"},{"id":239,"details":{"paperId":"443233ef16ea4d9fc5962cd8a9fc4cd0bd4f6dc0","externalIds":{"MAG":"2168204294","DOI":"10.1099/VIR.0.19769-0","CorpusId":"13184656","PubMed":"15105532"},"title":"The small hydrophobic (SH) protein accumulates within lipid-raft structures of the Golgi complex during respiratory syncytial virus infection.","abstract":"The cellular distribution of the small hydrophobic (SH) protein in respiratory syncytial virus (RSV)-infected cells was examined. Although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate in the Golgi complex within membrane structures that were enriched in the raft lipid, GM1. The ability of the SH protein to interact with lipid-raft membranes was further confirmed by examining its detergent-solubility properties in Triton X-100 at 4 degrees C. This analysis showed that a large proportion of the SH protein exhibited detergent-solubility characteristics that were consistent with an association with lipid-raft membranes. Analysis of virus-infected cells by immuno-transmission electron microscopy revealed SH protein clusters on the cell surface, but only very low levels of the protein appeared to be associated with mature virus filaments and inclusion bodies. These data suggest that during virus infection, the compartments in the secretory pathway, such as the endoplasmic reticulum (ER) and Golgi complex, are major sites of accumulation of the SH protein. Furthermore, although a significant amount of this protein interacts with lipid-raft membranes within the Golgi complex, its presence within mature virus filaments is minimal.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The data suggest that during virus infection, the compartments in the secretory pathway, such as the endoplasmic reticulum (ER) and Golgi complex, are major sites of accumulation of the SH protein."}},"tag":"EXP"},{"id":346,"details":{"paperId":"806564e12c43ecaafef0c531e8fce0a0b45a4d19","externalIds":{"MAG":"1573016254","DOI":"10.1128/jvi.70.9.6143-6150.1996","CorpusId":"40106863","PubMed":"8709239"},"title":"The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome","abstract":"The first nine genes of respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus, are separated by intergenic regions which range in size from 1 to 52 nucleotides for strain A2 and lack obvious consensus elements except that each ends in an A (genome sense). Their significance for gene expression was investigated by using RSV-CAT-LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV genomic RNA in which the viral genes were replaced by a negative-sense copy of the translational open reading frame (ORF) encoding chloramphenicol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUC) as the downstream gene. These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals and separated by the naturally occurring G/F intergenic region. The RSV-CAT-LUC minigenome was synthesized in vitro and transfected into RSV-infected cells, and synthesis of the CAT and LUC mRNAs was monitored by enzyme assay and Northern (RNA) blot hybridization. Surprisingly, substitution of each of the other naturally occurring RSV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs. Substitution of a nonnatural 10-nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurring intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRNAs would hybridize equally with a CAT-specific probe and their relative molar amounts could be determined. The level of expression of the downstream gene was 0.30 to 0.36 that of the upstream one. This determined the magnitude of RSV transcriptional polarity across a gene pair and confirmed that this value was very similar among the various intergenic regions. Minigenome transcription also yielded a CAT-LUC readthrough mRNA at a level 0.10 to 0.13 that of the LUC mRNA. In summary, the structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression and their significance for gene expression was investigated using RSV-CAT-LUC RNA."}},"tag":"EXP"},{"id":220,"details":{"paperId":"11d4996c8f9c2b695b9c92b4ddfe40cec94f8e0e","externalIds":{"MAG":"2114936988","DOI":"10.1099/0022-1317-74-3-485","CorpusId":"36336421","PubMed":"8445369"},"title":"The structure of the 5' terminal cap of the respiratory syncytial virus mRNA.","abstract":"The 5' terminal cap structure of the mRNAs of human respiratory syncytial virus synthesized in vitro in the presence of S-adenosyl-L-methionine consists of a 7-methyl guanosine linked to an unmethylated guanosine through a 5'-5' pyrophosphate linkage formed by using the alpha and beta phosphates of GTP. The complete cap structure is m7G (5')ppp(5')Gp ... which is devoid of ribose 2'-O-methylation. Capping, including methylation, is coupled to transcription. These results constitute the first report of a pneumoviral mRNA cap structure.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"These results constitute the first report of a pneumoviral mRNA cap structure that is devoid of ribose 2'-O-methylation and coupled to transcription."}},"tag":"EXP"},{"id":241,"details":{"paperId":"fd220a06e585893e61d5ccf69a22f6c0137b3747","externalIds":{"MAG":"2123408477","DOI":"10.1099/VIR.0.80318-0","CorpusId":"32514183","PubMed":"15557241"},"title":"Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism.","abstract":"Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein, and their thermostability before and after proteolytic cleavage was examined, finding them equally resistant to heat denaturation."}},"tag":"EXP"},{"id":168,"details":{"paperId":"96af8b1def844c3e3aac163017c550b3672b29d7","externalIds":{"MAG":"1996878801","DOI":"10.1073/PNAS.93.1.81","CorpusId":"2114909","PubMed":"8552680"},"title":"Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.","abstract":"RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded \"minigenome\" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is proposed that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions as well as in the case of a dicistronic minigenome, sequential transcription."}},"tag":"EXP"},{"id":337,"details":{"paperId":"095b59817da19139a2125323e127f487ae5f5f19","externalIds":{"MAG":"2171959150","DOI":"10.1128/jvi.66.11.6813-6818.1992","CorpusId":"19251716","PubMed":"1404620"},"title":"Transcription of human respiratory syncytial virus genome RNA in vitro: requirement of cellular factor(s)","abstract":"Extracts made from human respiratory syncytial virus (RSV)-infected Hep-2 cells synthesized mRNAs encoded by all known viral genes. In contrast, RSV ribonucleoproteins purified from infected cells failed to transcribe in vitro; transcription was restored by addition of a cytoplasmic extract of uninfected Hep-2 cells, demonstrating that a cellular factor(s) has a role in RSV gene expression. Quantitation of the individual gene mRNAs transcribed in vitro revealed polarity of transcription of the genome.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Extracts made from human respiratory syncytial virus-infected Hep-2 cells synthesized mRNAs encoded by all known viral genes, demonstrating that a cellular factor(s) has a role in RSV gene expression."}},"tag":"EXP"},{"id":24,"details":{"paperId":"3f6d637f7bfd3739fe5e69f454e0e91b6f4d08d0","externalIds":{"MAG":"2050147674","DOI":"10.1006/VIRO.2002.1541","CorpusId":"28342784","PubMed":"12350361"},"title":"Transcriptional termination modulated by nucleotides outside the characterized gene end sequence of respiratory syncytial virus.","abstract":"The genes of respiratory syncytial (RS) virus are transcribed sequentially by the viral RNA polymerase from a single 3'-proximal promoter. Polyadenylation and termination are directed by a sequence at the end of each gene, after which the polymerase crosses an intergenic region and reinitiates at the start sequence of the next gene. The 10 viral genes have different gene end sequences and different termination efficiencies, which allow for regulation of gene expression, since termination of each gene is required for initiation of the downstream gene. RNA sequences within the previously characterized 13 nucleotide gene end, including a conserved sequence 3'-UCAAU-5' and a tract of U residues, are important for termination. In this study, two additional sequence elements outside of the 13 nucleotide gene end were found to modulate termination efficiency: the A residue upstream of the 3'-UCAAU-5' sequence, and the first nucleotide of the intergenic region when it follows a U(4) tract.","publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Two additional sequence elements outside of the 13 nucleotide gene end were found to modulate termination efficiency: the A residue upstream of the 3'-UCAAU-5' sequence, and the first nucleotide of the intergenic region when it follows a U(4) tract."}},"tag":"EXP"},{"id":85,"details":{"paperId":"c2dd420c167fc1756730f4b219af290e0f20f463","externalIds":{"MAG":"1978461318","DOI":"10.1016/J.VIROL.2007.08.007","CorpusId":"25092555","PubMed":"17825340"},"title":"Ultrastructural analysis of the interaction between F-actin and respiratory syncytial virus during virus assembly.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is suggested that an intimate interaction occurs between actin and RSV, and that actin-associated signalling pathway, involving PI3K and Rac GTPase, may play an important role during virus assembly."}},"tag":"EXP"},{"id":254,"details":{"paperId":"c67d36be4db5ccb5423b8c95770a9edb78915230","externalIds":{"DOI":"10.1101/2022.11.28.517999","CorpusId":"254070918"},"title":"Ultrastructural characterization of a viral RNA and G-protein containing, membranous organelle formed in respiratory syncytial virus infected cells","abstract":"Respiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants and the elderly. In common with most viruses that replicate in the host cell cytoplasm, RSV induces the formation of cytoplasmic compartments within infected cells to sequester replicative processes from host countermeasures. The best characterised organelle formed during RSV infection is the inclusion body – the primary site of viral RNA synthesis - thought to form as a membrane-less biomolecular condensate. Fluorescence microscopy of cellular compartments using probes directed at the structural proteins of RSV and the intergenic regions of the RSV genome have identified a second class of organelles termed assembly granules. Here we use correlative microscopy to identify assembly granules in the cytoplasm of frozen hydrated RSV infected cells for imaging using cryogenic soft X-ray tomography and cryogenic electron tomography. We show that these compartments are membrane bound, enclosing large numbers of vesicles, some of which contain RSV ribonucleoprotein complexes. Further we show that these organelles are frequently adjacent to mitochondria and surrounded by ER-like membranes. We also observe vesicles connected by junctions suggesting mixing of contents and a mechanism for the different viral proteins to come together within the assembly granule prior to budding. Collectively, our data provides novel insights into the RSV assembly process.","publicationTypes":null,"tldr":{"model":"tldr@v2.0.0","text":"It is shown that assembly granules in the cytoplasm of frozen hydrated RSV infected cells for imaging using cryogenic soft X-ray tomography and cryogenic electron tomography are membrane bound, enclosing large numbers of vesicles, some of which contain RSV ribonucleoprotein complexes."}},"tag":"EXP"},{"id":248,"details":{"paperId":"df94bc6b1b6ffb096c2b54944aae16d7b2e2e0d2","externalIds":{"MAG":"2143452171","DOI":"10.1099/VIR.0.81786-0","CorpusId":"18143277","PubMed":"16760383"},"title":"Unravelling the complexities of respiratory syncytial virus RNA synthesis.","abstract":"Human respiratory syncytial virus (RSV) is the leading cause of paediatric respiratory disease and is the focus of antiviral- and vaccine-development programmes. These goals have been aided by an understanding of the virus genome architecture and the mechanisms by which it is expressed and replicated. RSV is a member of the order Mononegavirales and, as such, has a genome consisting of a single strand of negative-sense RNA. At first glance, transcription and genome replication appear straightforward, requiring self-contained promoter regions at the 3' ends of the genome and antigenome RNAs, short cis-acting elements flanking each of the genes and one polymerase. However, from these minimal elements, the virus is able to generate an array of capped, methylated and polyadenylated mRNAs and encapsidated antigenome and genome RNAs, all in the appropriate ratios to facilitate virus replication. The apparent simplicity of genome expression and replication is a consequence of considerable complexity in the polymerase structure and its cognate cis-acting sequences; here, our understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed.","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The understanding of mechanisms by which the RSV polymerase proteins interact with signals in the RNA template to produce different RNA products is reviewed."}},"tag":"EXP"},{"id":140,"details":{"paperId":"f0b438a04a79eeacde53e62fc7ea89f7a8c0fc42","externalIds":{"MAG":"2980304467","PubMedCentral":"6785563","DOI":"10.1038/s41467-019-12504-y","CorpusId":"203929677","PubMed":"31597913"},"title":"Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"It is demonstrated that viral m6A methylation upregulates RSV replication and pathogenesis and is identified as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses."}},"tag":"EXP"},{"id":305,"details":{"paperId":"00bab7496f80251b9e22237b0329e9b89445a35e","externalIds":{"MAG":"2106701493","DOI":"10.1128/JVI.01625-07","CorpusId":"14418884","PubMed":"17928346"},"title":"Viral and Host Factors in Human Respiratory Syncytial Virus Pathogenesis","abstract":"Human respiratory syncytial virus (RSV) was first isolated in 1956 from a laboratory chimpanzee with upper respiratory tract disease (for general reviews, see references [21][1], [57][2], [102][3], and [145][4]). RSV was quickly determined to be of human origin and was shown to be the leading","publicationTypes":["Review","JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Human respiratory syncytial virus (RSV) was first isolated in 1956 from a laboratory chimpanzee with upper respiratory tract disease and was quickly determined to be of human origin and was shown to be the leading virus."}},"tag":"EXP"},{"id":97,"details":{"paperId":"439c732fa48ab408e5769a5a3c23342abc667b82","externalIds":{"DOI":"10.1016/j.virol.2021.02.008","CorpusId":"232141984","PubMed":"33677389"},"title":"Virus-induced activation of the rac1 protein at the site of respiratory syncytial virus assembly is a requirement for virus particle assembly on infected cells.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"Evidence is provided that the increased rac1 activation occurred at the site of RSV assembly and facilitated F-actin remodeling during virus morphogenesis and provided evidence that the virus envelope proteins are trafficked to membrane microdomains containing the rac1 protein."}},"tag":"EXP"},{"id":57,"details":{"paperId":"bf31b780c2ae4265e03a1d2729421e96b270830a","externalIds":{"MAG":"2569496044","DOI":"10.1016/j.bbrc.2017.01.005","CorpusId":"29048597","PubMed":"28062184"},"title":"c-Jun N-terminal kinase activity is required for efficient respiratory syncytial virus production.","abstract":null,"publicationTypes":["JournalArticle"],"tldr":{"model":"tldr@v2.0.0","text":"The results implicate JNK1/2 as a key host factor for RSV virus production, raising the possibility of agents targeting JNK activity as potential anti-RSV therapeutics."}},"tag":"EXP"}]