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 The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner

2009 ◽  
Vol 83 (13) ◽  
pp. 6363-6374 ◽  
Author(s):  
Thi-Lan Tran ◽  
Nathalie Castagné ◽  
Virginie Dubosclard ◽  
Sylvie Noinville ◽  
Emmanuelle Koch ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Rna Binding ◽  
Amino Acid Residues ◽  
Polymerase Complex ◽  
Bacterial Rna ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Α Helix ◽  
Zinc Binding Domain

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.

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 Respiratory syncytial virus assembly occurs in GM1-rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin-1

2002 ◽  
Vol 83 (8) ◽  
pp. 1841-1850 ◽  
Author(s):  
Gaie Brown ◽  
Helen W. McL. Rixon ◽  
Richard J. Sugrue
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Surface ◽  
Host Cell ◽  
Lipid Raft ◽  
Cellular Distribution ◽  
Assembly Process ◽  
Caveolin 1 ◽  
Cytoplasmic Vesicles ◽  
Infected Cells ◽  
Syncytial Virus

We have previously shown that respiratory syncytial virus (RSV) assembly occurs within regions of the host-cell surface membrane that are enriched in the protein caveolin-1 (cav-1). In this report, we have employed immunofluorescence microscopy to further examine the RSV assembly process. Our results show that RSV matures at regions of the cell surface that, in addition to cav-1, are enriched in the lipid-raft ganglioside GM1. Furthermore, a comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the cellular distribution of phosphocaveolin-1 (pcav-1). In mock-infected cells, pcav-1 was located at regions of the cell that interact with the extracellular matrix, termed focal adhesions (FA). In contrast, RSV-infected cells showed both a decrease in the levels of pcav-1 associated with FA and the appearance of pcav-1-containing cytoplasmic vesicles, the latter being absent in mock-infected cells. These cytoplasmic vesicles were clearly visible between 9 and 18 h post-infection and coincided with the formation of RSV filaments, although we did not observe a direct association of pcav-1 with mature virus. In addition, we noted a strong colocalization between pcav-1 and growth hormone receptor binding protein-7 (Grb7), within these cytoplasmic vesicles, which was not observed in mock-infected cells. Collectively, these findings show that the RSV assembly process occurs within specialized lipid-raft structures on the host-cell plasma membrane, induces the cellular redistribution of pcav-1 and results in the formation of cytoplasmic vesicles that contain both pcav-1 and Grb7.

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 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.

Association of matrix protein of respiratory syncytial virus with the host cell membrane of infected cells

2003 ◽  
Vol 149 (1) ◽  
pp. 199-210 ◽  
Author(s):  
A. Marty ◽  
J. Meanger ◽  
J. Mills ◽  
B. Shields ◽  
R. Ghildyal
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Membrane ◽  
Host Cell ◽  
Matrix Protein ◽  
Host Cell Membrane ◽  
Infected Cells ◽  
Syncytial Virus

Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus

2001 ◽  
Vol 114 (15) ◽  
pp. 2747-2754
Author(s):  
David G. Swan ◽  
Rowena Stern ◽  
Sue McKellar ◽  
Kirsten Phillips ◽  
Chris A. L. Oura ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Cell ◽  
Cell Lines ◽  
Theileria Annulata ◽  
Open Reading Frames ◽  
Nuclear Extracts ◽  
Genes Encoding ◽  
Cell Gene Expression ◽  
Infected Cells ◽  
Host Nucleus

Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.

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