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 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 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

scholarly journals Biophysical and Dynamic Characterization of Fine-Tuned Binding of the Human Respiratory Syncytial Virus M2-1 Core Domain to Long RNAs

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Icaro P. Caruso ◽  
Giovana C. Guimarães ◽  
Vitor B. Machado ◽  
Marcelo A. Fossey ◽  
Dieter Willbold ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Conformational Changes ◽  
Rna Binding ◽  
Contact Region ◽  
Human Respiratory Syncytial Virus ◽  
Dynamic Characterization ◽  
Core Domain ◽  
Syncytial Virus ◽  
Rna Complex

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.

scholarly journals Stable Attenuation of Human Respiratory Syncytial Virus for Live Vaccines by Deletion and Insertion of Amino Acids in the Hinge Region between the mRNA Capping and Methyltransferase Domains of the Large Polymerase Protein

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Miaoge Xue ◽  
Rongzhang Wang ◽  
Olivia Harder ◽  
Phylip Chen ◽  
Mijia Lu ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Human Respiratory Syncytial Virus ◽  
Hinge Region ◽  
Vaccine Candidates ◽  
Recombinant Viruses ◽  
L Protein ◽  
Double Deletion ◽  
Mrna Capping ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (RSV) is the leading viral cause of lower respiratory tract disease in infants and children worldwide. Currently, there are no FDA-approved vaccines to combat this virus. The large (L) polymerase protein of RSV replicates the viral genome and transcribes viral mRNAs. The L protein is organized as a core ring-like domain containing the RNA-dependent RNA polymerase and an appendage of globular domains containing an mRNA capping region and a cap methyltransferase region, which are linked by a flexible hinge region. Here, we found that the flexible hinge region of RSV L protein is tolerant to amino acid deletion or insertion. Recombinant RSVs carrying a single or double deletion or a single alanine insertion were genetically stable, highly attenuated in immortalized cells, had defects in replication and spread, and had a delay in innate immune cytokine responses in primary, well-differentiated, human bronchial epithelial (HBE) cultures. The replication of these recombinant viruses was highly attenuated in the upper and lower respiratory tracts of cotton rats. Importantly, these recombinant viruses elicited high levels of neutralizing antibody and provided complete protection against RSV replication. Taken together, amino acid deletions or insertions in the hinge region of the L protein can serve as a novel approach to rationally design genetically stable, highly attenuated, and immunogenic live virus vaccine candidates for RSV. IMPORTANCE Despite tremendous efforts, there are no FDA-approved vaccines for human respiratory syncytial virus (RSV). A live attenuated RSV vaccine is one of the most promising vaccine strategies for RSV. However, it has been a challenge to identify an RSV vaccine strain that has an optimal balance between attenuation and immunogenicity. In this study, we generated a panel of recombinant RSVs carrying a single and double deletion or a single alanine insertion in the large (L) polymerase protein that are genetically stable, sufficiently attenuated, and grow to high titer in cultured cells, while retaining high immunogenicity. Thus, these recombinant viruses may be promising vaccine candidates for RSV.

scholarly journals Complete Genome Sequences of 13 Human Respiratory Syncytial Virus Subgroup A Strains of Genotypes NA1 and ON1 Isolated in the Philippines

2018 ◽  
Vol 6 (10) ◽  
Author(s):  
Rungnapa Malasao ◽  
Yuki Furuse ◽  
Michiko Okamoto ◽  
Clyde Dapat ◽  
Mayuko Saito ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Complete Genome ◽  
Acute Respiratory Infection ◽  
The Philippines ◽  
Human Respiratory Syncytial Virus ◽  
Genome Sequences ◽  
L Proteins ◽  
Syncytial Virus ◽  
Virus Strains

ABSTRACT Complete genome sequences of 13 human respiratory syncytial virus strains were determined from samples obtained from children hospitalized in the Philippines between 2012 and 2013 because of acute respiratory infection. We identified amino acid polymorphisms between the NA1 and ON1 genotypes in the P, G, F, and L proteins.

scholarly journals Complete Genome Sequences of 12 Human Respiratory Syncytial Virus (Human Orthopneumovirus) Strains Detected in Children with Repeated Subgroup B Infections in the Philippines

2018 ◽  
Vol 7 (22) ◽  
Author(s):  
Michiko Okamoto ◽  
Masahiro Sakamoto ◽  
Clyde Dapat ◽  
Mayuko Saito ◽  
Mariko Saito-Obata ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Complete Genome ◽  
The Philippines ◽  
Human Respiratory Syncytial Virus ◽  
Genome Sequences ◽  
Common Amino Acid ◽  
L Proteins ◽  
Syncytial Virus ◽  
Virus Strains

Complete genome sequences were determined for 12 human respiratory syncytial virus strains collected from nasopharyngeal samples obtained from children with repeated subgroup B infections. Eight common amino acid polymorphisms in the G, F, and L proteins were identified between the viruses detected in initial and subsequent infections.

scholarly journals Genetic Characterization of Fusion Protein of Human Respiratory Syncytial Virus: Beijing

2012 ◽  
Vol 1 (2) ◽  
pp. 74-79
Author(s):  
Qi Lu ◽  
Chun-xia Zhao ◽  
Kun-ling Shen ◽  
Wen-bo Xu ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Fusion Protein ◽  
Human Respiratory Syncytial Virus ◽  
Genetic Characteristics ◽  
Sequence Identity ◽  
Pcr Products ◽  
A Subunit ◽  
Syncytial Virus ◽  
Tract Infections

Abstract Objective Fusion protein is a subunit of the human respiratory syncytial virus (HRSV) and a potential vaccine candidate. Thus, a study on the genetic characteristics of F protein was considered important for further investigations in this field. The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide. Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing. Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs. Results Seventy-six samples (23.1%) were positive for HRSV. The percentage of cases in patients younger than 1 year was 84.21%. Among the six Beijing isolates, four belonged to subgroup A, whose respective F genes shared 97.0%-97.4% nucleotide sequence identity and 92.1%-93.0% amino acid sequence identity. The other two isolates belonged to subgroup B. Here, 97.3% and 98.2% sequence identity were found at nucleotide and amino acid levels, respectively. Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other, but those two within subgroup B distributed in two distinct clusters. Subgroup A and B strains co-circulated, indicating that two different transmission chains occurred in Beijing from 2003-2004.

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