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 Biophysical and dynamic characterization of a fine-tuned binding of the human Respiratory Syncytial Virus M2-1 core domain to long RNAs

2020 ◽  
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

ABSTRACTThe 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.IMPORTANCEThe 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.

scholarly journals Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Normand Blais ◽  
Martin Gagné ◽  
Yoshitomo Hamuro ◽  
Patrick Rheault ◽  
Martine Boyer ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antibody Response ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Human Respiratory Syncytial Virus ◽  
Vaccine Antigen ◽  
Significant Advance ◽  
F Protein ◽  
Syncytial Virus

ABSTRACT The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen. IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F) glycoprotein is considered one of the most promising vaccine candidates, and recent evidences suggest that the prefusion (pre-F) state is a superior target for neutralizing antibodies. Our study presents the physicochemical characterization of Pre-F-GCN4t, a molecule designed to be stabilized in the pre-F conformation. To confirm its pre-F conformation, Pre-F-GCN4t was analyzed in parallel with Post-F-XC, a molecule in the post-F conformation. Our results show that Pre-F-GCN4t presents characteristics of a stabilized pre-F conformation and support its use as an RSV vaccine antigen. Such an antigen may represent a significant advance in the development of an RSV vaccine.

Molecular characterization of the glycoprotein and fusion protein in human respiratory syncytial virus subgroup A: Emergence of ON-1 genotype in Iran

2019 ◽  
Vol 71 ◽  
pp. 166-178 ◽  
Author(s):  
Somayeh Shatizadeh Malekshahi ◽  
Shaghayegh Razaghipour ◽  
Yazdan Samieipoor ◽  
Farhad B. Hashemi ◽  
Ali Akbar Rahbari Manesh ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Molecular Characterization ◽  
Human Respiratory Syncytial Virus ◽  
Syncytial Virus

Clinical characterization of influenza A and human respiratory syncytial virus among patients with influenza like illness

2016 ◽  
Vol 89 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Swati Saxena ◽  
Dharamveer Singh ◽  
Amreen Zia ◽  
Jyoti Umrao ◽  
Naveen Srivastava ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Influenza A ◽  
Human Respiratory Syncytial Virus ◽  
Influenza Like Illness ◽  
Syncytial Virus

scholarly journals Sequence elements of the fusion peptide of human respiratory syncytial virus fusion protein required for activity

2006 ◽  
Vol 87 (6) ◽  
pp. 1649-1658 ◽  
Author(s):  
Diana Martín ◽  
Lesley J. Calder ◽  
Blanca García-Barreno ◽  
John J. Skehel ◽  
José A. Melero
Keyword(s):  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Fusion Peptide ◽  
Human Respiratory Syncytial Virus ◽  
Full Length ◽  
Fusion Peptides ◽  
Sequence Elements ◽  
Syncytial Virus ◽  
Simple Molecules

We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein () representing the ectodomain of the full-length F. molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the 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 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 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 , and these mutants remained as unaggregated cones after cleavage. In contrast, more conservative changes did not inhibit the change of shape and aggregation of . When the same changes were introduced in the fusion peptide of full-length F, only the mutations that inhibited aggregation of prevented membrane fusion. Thus, the conformational changes that follow completion of cleavage of the 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.

scholarly journals Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches

2007 ◽  
Vol 88 (10) ◽  
pp. 2719-2723 ◽  
Author(s):  
Sheng-Jiun Wu ◽  
Albert Schmidt ◽  
Eric J. Beil ◽  
Nicole D. Day ◽  
Patrick J. Branigan ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antigenic Site ◽  
Neutralizing Antibody ◽  
Human Respiratory Syncytial Virus ◽  
Peptide Sequence ◽  
F Protein ◽  
Syncytial Virus ◽  
A Minor ◽  
Key Residues

Chimeric 101F (ch101F) is a mouse–human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422–438 spanning antigenic site IV, V, VI was analysed. Residues 423–436 comprise the minimal peptide sequence for ch101F binding. Substitution analysis revealed that R429 and K433 are critical for ch101F binding, whilst K427 makes a minor contribution. Binding of ch101F to a series of single mutations at positions 427, 429 and 433 in the F protein expressed recombinantly on the cell surface confirmed the peptide results. Sequence analysis of viruses selected for resistance to neutralization by ch101F indicated that a single change (K433T) in the F protein allowed ch101F escape. The results confirm that ch101F and palivizumab have different epitope specificity and define key residues for ch101F recognition.

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