scholarly journals Neutralization of Human Respiratory Syncytial Virus Infectivity by Antibodies and Low-Molecular-Weight Compounds Targeted against the Fusion Glycoprotein

2010 ◽  
Vol 84 (16) ◽  
pp. 7970-7982 ◽  
Author(s):  
Margarita Magro ◽  
David Andreu ◽  
Paulino Gómez-Puertas ◽  
José A. Melero ◽  
Concepción Palomo
Keyword(s):  
Molecular Weight ◽  
Respiratory Syncytial Virus ◽  
Vaccine Development ◽  
Human Respiratory Syncytial Virus ◽  
Heptad Repeat ◽  
Low Molecular Weight ◽  
Virus Infectivity ◽  
F Protein ◽  
Low Molecular Weight Compounds ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (HRSV) fusion (F) protein is an essential component of the virus envelope that mediates fusion of the viral and cell membranes, and, therefore, it is an attractive target for drug and vaccine development. Our aim was to analyze the neutralizing mechanism of anti-F antibodies in comparison with other low-molecular-weight compounds targeted against the F molecule. It was found that neutralization by anti-F antibodies is related to epitope specificity. Thus, neutralizing and nonneutralizing antibodies could bind equally well to virions and remained bound after ultracentrifugation of the virus, but only the former inhibited virus infectivity. Neutralization by antibodies correlated with inhibition of cell-cell fusion in a syncytium formation assay, but not with inhibition of virus binding to cells. In contrast, a peptide (residues 478 to 516 of F protein [F478-516]) derived from the F protein heptad repeat B (HRB) or the organic compound BMS-433771 did not interfere with virus infectivity if incubated with virus before ultracentrifugation or during adsorption of virus to cells at 4°C. These inhibitors must be present during virus entry to effect HRSV neutralization. These results are best interpreted by asserting that neutralizing antibodies bind to the F protein in virions interfering with its activation for fusion. Binding of nonneutralizing antibodies is not enough to block this step. In contrast, the peptide F478-516 or BMS-433771 must bind to F protein intermediates generated during virus-cell membrane fusion, blocking further development of this process.

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.

scholarly journals Local Evolutionary Patterns of Human Respiratory Syncytial Virus Derived from Whole-Genome Sequencing

2015 ◽  
Vol 89 (7) ◽  
pp. 3444-3454 ◽  
Author(s):  
Charles N. Agoti ◽  
James R. Otieno ◽  
Patrick K. Munywoki ◽  
Alexander G. Mwihuri ◽  
Patricia A. Cane ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Large Scale ◽  
Vaccine Development ◽  
Human Respiratory Syncytial Virus ◽  
Genomic Variation ◽  
Genomic Sequences ◽  
The Novel ◽  
Sequencing Method ◽  
The World ◽  
Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies.IMPORTANCEThe new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

scholarly journals RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function

2013 ◽  
Vol 94 (8) ◽  
pp. 1691-1700 ◽  
Author(s):  
Jane Tian ◽  
Kelly Huang ◽  
Subramaniam Krishnan ◽  
Catherine Svabek ◽  
Daniel C. Rowe ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Epithelial Cells ◽  
Syncytium Formation ◽  
Human Respiratory Syncytial Virus ◽  
Host Cells ◽  
Type I ◽  
F Protein ◽  
Viral Spread ◽  
Infected Cells ◽  
Syncytial Virus

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

scholarly journals Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

Virology ◽  
2010 ◽  
Vol 396 (2) ◽  
pp. 226-237 ◽  
Author(s):  
Abdul S. Yunus ◽  
Trent P. Jackson ◽  
Katherine Crisafi ◽  
Irina Burimski ◽  
Nicole R. Kilgore ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Elevated Temperature ◽  
Human Respiratory Syncytial Virus ◽  
F Protein ◽  
Helix Bundle ◽  
Syncytial Virus

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.

scholarly journals The Cytoplasmic Tail of the Human Respiratory Syncytial Virus F Protein Plays Critical Roles in Cellular Localization of the F Protein and Infectious Progeny Production

2006 ◽  
Vol 80 (21) ◽  
pp. 10465-10477 ◽  
Author(s):  
Antonius G. P. Oomens ◽  
Kevin P. Bevis ◽  
Gail W. Wertz
Keyword(s):  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Lipid Rafts ◽  
Cellular Localization ◽  
Cytoplasmic Tail ◽  
Human Respiratory Syncytial Virus ◽  
Viral Glycoprotein ◽  
Protein Modifications ◽  
F Protein ◽  
Syncytial Virus

ABSTRACT The importance of the F protein cytoplasmic tail (CT) for replication of human respiratory syncytial virus (HRSV) was examined by monitoring the behavior of viruses expressing F proteins with a modified COOH terminus. The F protein mutant viruses were recovered and amplified under conditions where F protein function was complemented by expression of a heterologous viral envelope protein. The effect of the F protein modifications was then examined in the context of a viral infection in standard cell types (Vero and HEp-2). The F protein modifications consisted of a deletion of the predicted CT or a replacement of the CT with the CT of the vesicular stomatitis virus (VSV) G protein. In addition, engineered HRSVs that lacked all homologous glycoprotein genes (SH, G, and F) and expressed instead either the authentic VSV G protein or a VSV G containing the HRSV F protein CT were examined. We found that deletion or replacement of the F protein CT seriously impaired the production of infectious progeny. Cells infected with viruses bearing CT modifications displayed increased F protein surface expression and increased syncytium formation. The distribution of F protein in the plasma membrane of infected cells was altered, resulting in an F protein that was evenly distributed rather than localized predominantly to virus-induced surface filaments. CT deletion or exchange also abrogated interaction of F protein with Triton-insoluble lipid rafts. Addition of the F protein CT to the VSV G protein, expressed as the only viral glycoprotein in an HRSV genome, had the opposite effects: the number of infectious progeny was higher, the surface distribution was changed from relatively even to localized, and the proportion of VSV G protein associated with lipid rafts was higher. Together, these results show that the HRSV F protein CT plays a critical role in F protein cellular localization and production of infectious virus and suggest that the function provided by the CT is independent of the F protein ectodomain and transmembrane domain and is mediated by F protein-lipid raft interaction.

scholarly journals The cytoplasmic domain of the F protein of Human respiratory syncytial virus is not required for cell fusion

2006 ◽  
Vol 87 (2) ◽  
pp. 395-398 ◽  
Author(s):  
Patrick J. Branigan ◽  
Nicole D. Day ◽  
Changbao Liu ◽  
Lester L. Gutshall ◽  
José A. Melero ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Fusion ◽  
Fusion Proteins ◽  
Cytoplasmic Domain ◽  
Cysteine Residue ◽  
Human Respiratory Syncytial Virus ◽  
Cytoplasmic Domains ◽  
F Protein ◽  
Avian Pneumovirus ◽  
Syncytial Virus

The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.

scholarly journals Distinct gene subsets are induced at different time points after human respiratory syncytial virus infection of A549 cells

2007 ◽  
Vol 88 (2) ◽  
pp. 570-581 ◽  
Author(s):  
Isidoro Martínez ◽  
Luis Lombardía ◽  
Blanca García-Barreno ◽  
Orlando Domínguez ◽  
José A. Melero
Keyword(s):  
Respiratory Syncytial Virus ◽  
Time Course ◽  
A549 Cells ◽  
Cellular Protein ◽  
Human Respiratory Syncytial Virus ◽  
Virus Infectivity ◽  
Dependent Manner ◽  
Down Regulation ◽  
Cytoskeleton Organization ◽  
Syncytial Virus

cDNA microarray technology was applied to time course analysis of differentially expressed genes in A549 cells following human respiratory syncytial virus (HRSV) infection. Both up- and down-regulation of cellular genes were observed in a time-dependent manner. However, gene up-regulation prevailed over gene down-regulation. Virus infectivity was required as UV-inactivated virus failed to up-regulate/down-regulate those genes. At early times post-infection (0–6 h p.i.) 85 genes were up-regulated. Some of those genes were involved in cell growth/proliferation, cellular protein metabolism and cytoskeleton organization. Among the most strongly up-regulated genes at that time were the urokinase plasminogen activator (PLAU) and its receptor (PLAUR), a pleiotropic system involved in many biological processes, including chemotaxis and inflammation. Functionally related genes encoding the α- and β-chains of several integrins were also up-regulated within the first 12 h of infection. Genes up-regulated between 6 and 12 h p.i. included interferon-stimulated genes (ISGs), genes related to oxidative stress and genes of the non-canonical NF-κB pathway. At later times, genes involved in the immune response became predominant among the up-regulated genes, most of them being ISGs. Different up-regulation kinetics of cytokine and cytokine-signalling-related genes were also observed. These results highlight the dynamic interplay between the virus and the host cell and provide a general picture of changes in cellular gene expression along the HRSV replicative cycle.

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