scholarly journals Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 535 ◽  
Author(s):  
Shadi Shahriari ◽  
Ke-jun Wei ◽  
Reena Ghildyal
Keyword(s):  
Cell Culture ◽  
Respiratory Syncytial Virus ◽  
Cytochalasin D ◽  
Full Length ◽  
Host Protein ◽  
M Protein ◽  
Apical Surface ◽  
Infected Cells ◽  
Syncytial Virus

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.

scholarly journals In Vitro and In Vivo Fitness of Respiratory Syncytial Virus Monoclonal Antibody Escape Mutants

2006 ◽  
Vol 80 (23) ◽  
pp. 11651-11657 ◽  
Author(s):  
Xiaodong Zhao ◽  
Enmei Liu ◽  
Fu-Ping Chen ◽  
Wayne M. Sullender
Keyword(s):  
Cell Culture ◽  
Monoclonal Antibody ◽  
Respiratory Syncytial Virus ◽  
Viral Population ◽  
Nucleotide Substitutions ◽  
Cotton Rats ◽  
Syncytial Virus ◽  
In Vivo Growth

ABSTRACT Respiratory syncytial virus (RSV) is the only infectious disease for which a monoclonal antibody (MAb) is used in humans. Palivizumab (PZ) is a humanized murine MAb to the F protein of RSV. PZ-resistant viruses appear after in vitro and in vivo growth of RSV in the presence of PZ. Fitness for replication could be a determinant of the likelihood of dissemination of resistant viruses. We assessed the fitness of two PZ-resistant viruses (F212 and MP4). F212 grew less well in cell culture than the parent A2 virus and was predicted to be less fit than A2. Equal amounts of F212 and A2 were mixed and passaged in cell culture. F212 disappeared from the viral population, indicating it was less fit than the A2 virus. The MP4 virus grew as well as A2 in culture and in cotton rats. A2/MP4 virus input ratios of 1:1, 10:1, 100:1, and 1,000:1 were compared in competitive replication. For all input ratios except 1,000:1, the MP4 virus became dominant, supplanting the A2 virus. The MP4 virus also dominated the A2 virus during growth in cotton rats. Thus, the mutant MP4 virus was more fit than A2 virus in both in vitro and in vivo competitive replication. Whether this fitness difference was due to the identified nucleotide substitutions in the F gene or to mutations elsewhere in the genome is unknown. Understanding the mechanisms by which mutant virus fitness increased or decreased could prove useful for consideration in attenuated vaccine design efforts.

scholarly journals The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

2002 ◽  
Vol 76 (21) ◽  
pp. 10776-10784 ◽  
Author(s):  
Bin Lu ◽  
Chien-Hui Ma ◽  
Robert Brazas ◽  
Hong Jin
Keyword(s):  
Respiratory Syncytial Virus ◽  
Virus Replication ◽  
Vero Cells ◽  
Phosphorylation Sites ◽  
N Protein ◽  
P Protein ◽  
Infected Cells ◽  
Syncytial Virus

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.

scholarly journals Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

2020 ◽  
pp. 7-14
Author(s):  
N. A. Demidova ◽  
R. R. Klimova ◽  
A. A. Kushch ◽  
E. I. Lesnova ◽  
O. V. Masalova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rsv Infection ◽  
Hybridoma Technology ◽  
Infected Cells ◽  
Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

scholarly journals Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity In Vitro

1998 ◽  
Vol 72 (9) ◽  
pp. 7221-7227 ◽  
Author(s):  
C. Bourgeois ◽  
J. B. Bour ◽  
K. Lidholt ◽  
C. Gauthray ◽  
P. Pothier
Keyword(s):  
Respiratory Syncytial Virus ◽  
Heparan Sulfate ◽  
G Protein ◽  
Enzymatic Digestion ◽  
Virus Infectivity ◽  
Rsv Infection ◽  
Infected Cells ◽  
Virus Culture ◽  
Syncytial Virus

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.

scholarly journals The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism

2009 ◽  
Vol 83 (11) ◽  
pp. 5353-5362 ◽  
Author(s):  
Reena Ghildyal ◽  
Adeline Ho ◽  
Manisha Dias ◽  
Lydia Soegiyono ◽  
Phillip G. Bardin ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Nuclear Export Signal ◽  
M Protein ◽  
Nuclear Accumulation ◽  
Infected Cells ◽  
Syncytial Virus ◽  
In Cells

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.

scholarly journals Verdinexor (KPT-335), a Selective Inhibitor of Nuclear Export, Reduces Respiratory Syncytial Virus Replication In Vitro

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Patricia A. Jorquera ◽  
Cynthia Mathew ◽  
Jennifer Pickens ◽  
Colin Williams ◽  
Jasmina M. Luczo ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Young Children ◽  
Nuclear Export ◽  
Symptomatic Relief ◽  
Antiviral Drugs ◽  
M Protein ◽  
Nuclear Accumulation ◽  
Syncytial Virus ◽  
Infants And Young Children

ABSTRACT Respiratory syncytial virus (RSV) is a leading cause of hospitalization of infants and young children, causing considerable respiratory disease and repeat infections that may lead to chronic respiratory conditions such as asthma, wheezing, and bronchitis. RSV causes ∼34 million new episodes of lower respiratory tract illness (LRTI) in children younger than 5 years of age, with >3 million hospitalizations due to severe RSV-associated LRTI. The standard of care is limited to symptomatic relief as there are no approved vaccines and few effective antiviral drugs; thus, a safe and efficacious RSV therapeutic is needed. Therapeutic targeting of host proteins hijacked by RSV to facilitate replication is a promising antiviral strategy as targeting the host reduces the likelihood of developing drug resistance. The nuclear export of the RSV M protein, mediated by the nuclear export protein exportin 1 (XPO1), is crucial for RSV assembly and budding. Inhibition of RSV M protein export by leptomycin B correlated with reduced RSV replication in vitro. In this study, we evaluated the anti-RSV efficacy of Verdinexor (KPT-335), a small molecule designed to reversibly inhibit XPO1-mediated nuclear export. KPT-335 inhibited XPO1-mediated transport and reduced RSV replication in vitro. KPT-335 was effective against RSV A and B strains and reduced viral replication following prophylactic or therapeutic administration. Inhibition of RSV replication by KPT-335 was due to a combined effect of reduced XPO1 expression, disruption of the nuclear export of RSV M protein, and inactivation of the NF-κB signaling pathway. IMPORTANCE RSV is an important cause of LRTI in infants and young children for which there are no suitable antiviral drugs offered. We evaluated the efficacy of KPT-335 as an anti-RSV drug and show that KPT-335 inhibits XPO1-mediated nuclear export, leading to nuclear accumulation of RSV M protein and reduction in RSV levels. KPT-335 treatment also resulted in inhibition of proinflammatory pathways, which has important implications for its effectiveness in vivo.

scholarly journals Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2786
Author(s):  
Hong-Mei Li ◽  
Reena Ghildyal ◽  
Mengjie Hu ◽  
Kim C. Tran ◽  
Lora M. Starrs ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Dna Binding ◽  
Host Cell ◽  
Rna Binding ◽  
M Protein ◽  
Free System ◽  
Transcriptional Inhibition ◽  
Infected Cells ◽  
Syncytial Virus

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.

scholarly journals Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells

2002 ◽  
Vol 83 (4) ◽  
pp. 753-757 ◽  
Author(s):  
R. Ghildyal ◽  
J. Mills ◽  
M. Murray ◽  
N. Vardaxis ◽  
J. Meanger
Keyword(s):  
Respiratory Syncytial Virus ◽  
Matrix Protein ◽  
Rna Viruses ◽  
M Protein ◽  
Dependent Manner ◽  
Cytoplasmic Inclusions ◽  
Negative Strand ◽  
The Matrix ◽  
Infected Cells ◽  
Syncytial Virus

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.

scholarly journals Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene

2001 ◽  
Vol 75 (15) ◽  
pp. 6825-6834 ◽  
Author(s):  
Sunee Techaarpornkul ◽  
Naina Barretto ◽  
Mark E. Peeples
Keyword(s):  
Cell Culture ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Viral Entry ◽  
Target Cells ◽  
Virion Assembly ◽  
F Protein ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Small Hydrophobic

ABSTRACT Respiratory syncytial virus (RSV) produces three envelope glycoproteins, the attachment glycoprotein (G), the fusion (F) protein, and the small hydrophobic (SH) protein. It had been assumed, by analogy with other paramyxoviruses, that the G and F proteins would be required for the first two steps of viral entry, attachment and fusion. However, following repeated passage in cell culture, a viable mutant RSV that lacked both the G and SH genes was isolated (R. A. Karron, D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E. Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem, B. R. Murphy, and M. S. Sidhu, Proc. Natl. Acad. Sci. USA 94:13961–13966, 1997). To explore the roles of the G, F, and SH proteins in virion assembly, function, and cytopathology, we have modified the full-length RSV cDNA and used it to rescue infectious RSV lacking the G and/or SH genes. The three resulting viruses and the parental virus all contain the green fluorescent protein (GFP) gene that serves to identify infected cells. We have used purified, radiolabeled virions to examine virus production and function, in conjunction with GFP to quantify infected cells. We found that the G protein enhances virion binding to target cells but plays no role in penetration after attachment. The G protein also enhances cell-to-cell fusion, presumably via cell-to-cell binding, and enhances virion assembly or release. The presence or absence of the G protein in virions has no obvious effect on the content of F protein or host cell proteins in the virion. In growth curve experiments, the viruses lacking the G protein produced viral titers that were at least 10-fold lower than titers of viruses containing the G protein. This reduction is due in large part to the less efficient release of virions and the lower infectivity of the released virions. In the absence of the G protein, virus expressing both the F and SH proteins displayed somewhat smaller plaques, lower fusion activity, and slower viral entry than the virus expressing the F protein alone, suggesting that the SH protein has a negative effect on virus fusion in cell culture.

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