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 Structural Phosphoprotein M2-1 of the Human Respiratory Syncytial Virus Is an RNA Binding Protein

2000 ◽  
Vol 74 (21) ◽  
pp. 9858-9867 ◽  
Author(s):  
Isabel Cuesta ◽  
Xuehui Geng ◽  
Ana Asenjo ◽  
Nieves Villanueva
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Rna Binding ◽  
Protein Complexes ◽  
Binding Capacity ◽  
Human Respiratory Syncytial Virus ◽  
Sequence Specificity ◽  
Amino Acid Residues ◽  
Syncytial Virus ◽  
Rna Protein Complexes

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.

scholarly journals Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

Virology ◽  
2004 ◽  
Vol 330 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Terence P. McDonald ◽  
Andrew R. Pitt ◽  
Gaie Brown ◽  
Helen W. McL. Rixon ◽  
Richard J. Sugrue
Keyword(s):  
Respiratory Syncytial Virus ◽  
Lipid Rafts ◽  
Proteomic Analysis ◽  
Polymerase Complex ◽  
Infected Cells ◽  
Syncytial Virus

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 Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus

2009 ◽  
Vol 5 (1) ◽  
pp. e1000254 ◽  
Author(s):  
Viviane F. Botosso ◽  
Paolo M. de A. Zanotto ◽  
Mirthes Ueda ◽  
Eurico Arruda ◽  
Alfredo E. Gilio ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Positive Selection ◽  
G Protein ◽  
Protein Gene ◽  
Human Respiratory Syncytial Virus ◽  
Syncytial Virus

Amino Acid Variation within the Fusion Protein of Respiratory Syncytial Virus Subtype A and B Strains during Annual Epidemics in South Africa

Virus Genes ◽  
2005 ◽  
Vol 30 (2) ◽  
pp. 267-278 ◽  
Author(s):  
Elizabeth Agenbach ◽  
Caroline T. Tiemessen ◽  
Marietjie Venter
Keyword(s):  
South Africa ◽  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Fusion Protein ◽  
Amino Acid Variation ◽  
Syncytial Virus ◽  
Subtype A ◽  
Virus Subtype

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex

2020 ◽  
Vol 6 (10) ◽  
pp. 2800-2811
Author(s):  
Anand Balakrishnan ◽  
Edmund Price ◽  
Catherine Luu ◽  
Jacob Shaul ◽  
Charles Wartchow ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Biochemical Characterization ◽  
Rna Dependent Rna Polymerase ◽  
Polymerase Complex ◽  
Virus Rna ◽  
Syncytial Virus

scholarly journals Novel Variants of Respiratory Syncytial Virus A ON1 Associated With Increased Clinical Severity of Bronchiolitis

2020 ◽  
Vol 222 (1) ◽  
pp. 102-110
Author(s):  
Fabio Midulla ◽  
Greta Di Mattia ◽  
Raffaella Nenna ◽  
Carolina Scagnolari ◽  
Agnese Viscido ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Severity Score ◽  
Multinomial Logistic Regression ◽  
Clinical Severity ◽  
Term Infants ◽  
Clinical Severity Score ◽  
Glycosylation Sites ◽  
Novel Variants ◽  
Syncytial Virus

Abstract Background A study of respiratory syncytial virus-A (RSV A) genotype ON1 genetic variability and clinical severity in infants hospitalized with bronchiolitis over 6 epidemic seasons (2012–2013 to 2017–2018) was carried out. Methods From prospectively enrolled term infants hospitalized for bronchiolitis, samples positive for RSV A ON1 (N = 139) were sequenced in the second half of the G gene. Patients’ clinical data were obtained from medical files and each infant was assigned a clinical severity score. ANOVA comparison and adjusted multinomial logistic regression were used to evaluate clinical severity score and clinical parameters. Results The phylogenetic analysis of 54 strains showed 3 distinct clades; sequences in the last 2 seasons differed from previous seasons. The most divergent and numerous cluster of 2017–2018 strains was characterized by a novel pattern of amino acid changes, some in antigenic sites. Several amino acid changes altered predicted glycosylation sites, with acquisition of around 10 new O-glycosylation sites. Clinical severity of bronchiolitis increased in 2016–2017 and 2017–2018 and changed according to the epidemic seasons only. Conclusions Amino acid changes in the hypervariable part of G protein may have altered functions and/or changed its immunogenicity, leading to an impact on disease severity.

scholarly journals Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast.

1991 ◽  
Vol 174 (2) ◽  
pp. 425-434 ◽  
Author(s):  
K Falk ◽  
O Rötzschke ◽  
K Deres ◽  
J Metzger ◽  
G Jung ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Synthetic Peptides ◽  
Cell Epitope ◽  
Reversed Phase ◽  
T Cell Epitope ◽  
Amino Acid Residues ◽  
Allele Specific ◽  
Infected Cells ◽  
Natural Peptides

Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position.

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