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 The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

2004 ◽  
Vol 78 (1) ◽  
pp. 124-135 ◽  
Author(s):  
A. G. P. Oomens ◽  
Gail W. Wertz
Keyword(s):  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Low Ph ◽  
Human Respiratory Syncytial Virus ◽  
Dependent Manner ◽  
Practical Applications ◽  
Ph Dependent ◽  
Syncytial Virus

ABSTRACT Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP64/F, a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33°C than at 37°C. The open reading frames (ORFs) encoding GP64 or GP64/F, along with two marker ORFs encoding green fluorescent protein (GFP) and β-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP64/F protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSΔsh,g,f/GP64 or RSΔsh,g,f/GP64/F, respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSΔsh [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP64/F proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSΔsh,g,f/GP64 and RSΔsh,g,f/GP64/F replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

scholarly journals Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism

2004 ◽  
Vol 85 (12) ◽  
pp. 3677-3687 ◽  
Author(s):  
M. Begoña Ruiz-Argüello ◽  
Diana Martín ◽  
Steve A. Wharton ◽  
Lesley J. Calder ◽  
Steve R. Martín ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Human Respiratory Syncytial Virus ◽  
Virus Fusion ◽  
Before And After ◽  
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 Complete Genome Sequence of Human Respiratory Syncytial Virus from Shanghai, China

2015 ◽  
Vol 3 (5) ◽  
Author(s):  
Xuemin Fu ◽  
Yanwei Cheng ◽  
Zhixiang He ◽  
Wei Dong ◽  
Ke Lan ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Respiratory Syncytial Virus ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Respiratory Symptoms ◽  
Human Respiratory Syncytial Virus ◽  
Full Length ◽  
Group A ◽  
Syncytial Virus

We report here the complete genome sequence of human respiratory syncytial virus isolated from an outpatient child with fever and respiratory symptoms in Shanghai, China, in 2014. Phylogenetic analysis showed that the full-length respiratory syncytial virus (RSV) genome sequence belongs to human RSV (HRSV) group A.

scholarly journals Complete Genome Sequence of Human Respiratory Syncytial Virus from Lanzhou, China

2017 ◽  
Vol 5 (34) ◽  
Author(s):  
Chuanfeng Zhu ◽  
Shengfang Fu ◽  
Xv Zhou ◽  
Li Yu
Keyword(s):  
Phylogenetic Analysis ◽  
Respiratory Syncytial Virus ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Infectious Clone ◽  
Human Respiratory Syncytial Virus ◽  
Full Length ◽  
Full Length Cdna ◽  
Syncytial Virus

ABSTRACT A complete genome of human respiratory syncytial virus was sequenced and analyzed. Phylogenetic analysis showed that the full-length human respiratory syncytial virus (HRSV) genome sequence belongs to gene type NA1. We sequenced the genome in order to create the full-length cDNA infectious clone and develop vaccines against HRSV.

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.

Sign in / Sign up

Export Citation Format

Share Document