scholarly journals Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein

2015 ◽  
Vol 90 (1) ◽  
pp. 245-253 ◽  
Author(s):  
Jia Meng ◽  
Anne L. Hotard ◽  
Michael G. Currier ◽  
Sujin Lee ◽  
Christopher C. Stobart ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
G Protein ◽  
Clinical Isolate ◽  
Protein Function ◽  
Virus Binding ◽  
Attachment Protein ◽  
Recombinant Viruses ◽  
Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant virusesin vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection.IMPORTANCERSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable forin vitroreplication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, andin vitrogrowth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.

An in vitro selected binding protein (affibody) shows conformation-dependent recognition of the respiratory syncytial virus (RSV) G protein

1999 ◽  
Vol 4 (3-4) ◽  
pp. 237-252 ◽  
Author(s):  
Marianne Hansson ◽  
Jenny Ringdahl ◽  
Alain Robert ◽  
Ultan Power ◽  
Liliane Goetsch ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Binding Protein ◽  
Syncytial Virus

scholarly journals Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

2020 ◽  
pp. 7-14
Author(s):  
N. A. Demidova ◽  
R. R. Klimova ◽  
A. A. Kushch ◽  
E. I. Lesnova ◽  
O. V. Masalova ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rsv Infection ◽  
Hybridoma Technology ◽  
Infected Cells ◽  
Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

scholarly journals Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein

2015 ◽  
Vol 89 (16) ◽  
pp. 8258-8266 ◽  
Author(s):  
Anne L. Hotard ◽  
Elizabeth Laikhter ◽  
Kelsie Brooks ◽  
Tina V. Hartert ◽  
Martin L. Moore
Keyword(s):  
Respiratory Syncytial Virus ◽  
Buenos Aires ◽  
Virus Attachment ◽  
Recombinant Viruses ◽  
Fitness Advantage ◽  
Virus Life Cycle ◽  
Glycoprotein G ◽  
Syncytial Virus ◽  
Different Strains

ABSTRACTThere are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup, isolates are further divided into clades. Several years ago, multiple subgroup B isolates which contained a duplication of 60 nucleotides in the glycoprotein (G) gene were described. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene or a consensus BA G gene lacking the duplication (GΔdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed thatin vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness.IMPORTANCERespiratory syncytial virus (RSV) is an important pathogen for infants for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation.

scholarly journals Heparin-Like Structures on Respiratory Syncytial Virus Are Involved in Its Infectivity In Vitro

1998 ◽  
Vol 72 (9) ◽  
pp. 7221-7227 ◽  
Author(s):  
C. Bourgeois ◽  
J. B. Bour ◽  
K. Lidholt ◽  
C. Gauthray ◽  
P. Pothier
Keyword(s):  
Respiratory Syncytial Virus ◽  
Heparan Sulfate ◽  
G Protein ◽  
Enzymatic Digestion ◽  
Virus Infectivity ◽  
Rsv Infection ◽  
Infected Cells ◽  
Virus Culture ◽  
Syncytial Virus

ABSTRACT Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.

scholarly journals The Central Conserved Cystine Noose of the Attachment G Protein of Human Respiratory Syncytial Virus Is Not Required for Efficient Viral Infection In Vitro or In Vivo

2002 ◽  
Vol 76 (12) ◽  
pp. 6164-6171 ◽  
Author(s):  
Michael N. Teng ◽  
Peter L. Collins
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Respiratory Tract ◽  
G Protein ◽  
Virus Replication ◽  
Central Region ◽  
Syncytial Virus ◽  
Amino Acid Segment

ABSTRACT The G glycoprotein of human respiratory syncytial virus (RSV) was identified previously as the viral attachment protein. Although we and others recently showed that G is not essential for replication in vitro, it does affect the efficiency of replication in a cell type-dependent fashion and is required for efficient replication in vivo. The ectodomain of G is composed of two heavily glycosylated domains with mucin-like characteristics that are separated by a short central region that is relatively devoid of glycosylation sites. This central region contains a 13-amino acid segment that is conserved in the same form among RSV isolates and is overlapped by a second segment containing four cysteine residues whose spacings are conserved in the same form and which create a cystine noose. The conserved nature of the cystine noose and flanking 13-amino acid segment suggested that this region likely was important for attachment activity. To test this hypothesis, we constructed recombinant RSVs from which the region containing the cysteine residues was deleted together with part or all of the conserved 13-amino acid segment. Surprisingly, each deletion had little or no effect on the intracellular synthesis and processing of the G protein, the kinetics or efficiency of virus replication in vitro, or sensitivity to neutralization by soluble heparin in vitro. In addition, neither deletion had any discernible effect on the ability of RSV to infect the upper respiratory tract of mice and both resulted in a 3- to 10-fold reduction in the lower respiratory tract. Thus, although the G protein is necessary for efficient virus replication in vivo, this activity does not require the central conserved cystine noose region.

scholarly journals Neutralizing epitope of the Fusion Protein of Respiratory Syncytial Virus Embedded in the HA Molecule of LAIV Virus is not Sufficient to Prevent RS Virus Pulmonary Replication but Ameliorates Lung Pathology following RSV Infection in Mice

2020 ◽  
Vol 14 (1) ◽  
pp. 147-156
Author(s):  
Tatiana Kotomina ◽  
Irina Isakova-Sivak ◽  
Ekaterina Stepanova ◽  
Daria Mezhenskaya ◽  
Victoria Matyushenko ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Viral Vector ◽  
Replication Capacity ◽  
Lung Pathology ◽  
Neutralizing Epitope ◽  
Syncytial Virus ◽  
Chimeric Viruses

Aims: To develop experimental bivalent vaccines against influenza and RSV using a cold-adapted LAIV backbone. Background: Respiratory syncytial virus (RSV) is a causative agent of bronchiolitis and pneumonia in young children, elderly and immunocompromised adults. No vaccine against RSV has been licensed to date for various reasons. One of the promising platforms for designing RSV vaccine is the use of live attenuated influenza vaccine (LAIV) viruses to deliver RSV epitopes to the respiratory mucosa. Objective: To generate recombinant LAIV viruses encoding a neutralizing epitope of the RSV fusion protein and assess their protective potential against both influenza and RSV infections in a mouse model. Methods: Reverse genetics methods were used to rescue recombinant LAIV+HA/RSV viruses expressing chimeric hemagglutinins encoding the RSV-F epitope at its N-terminus using two different flexible linkers. BALB/c mice were intranasally immunized with two doses of the recombinant viruses and then challenged with the influenza virus or RSV. The LAIV viral vector and formalin-inactivated RSV (FI-RSV) were included as control vaccines. Protection was assessed by the reduction of virus pulmonary titers. In addition, RSV-induced lung pathology was evaluated by histopathology studies. Results: Two rescued chimeric LAIV+HA/RSV viruses were identical to the LAIV vector in terms of replication capacity in vitro and in vivo. The RSV-F neutralizing epitope was successfully expressed only if inserted into the HA molecule via G-linker, but not A-linker. Both chimeric viruses induced high influenza-specific antibody levels and fully protected mice against a lethal influenza challenge virus. However, they induced weak anti-RSV antibody responses which did not prevent RS virus replication upon challenge, and only LAIV-HA+G-RSV variant protected mice against RSV-induced lung pathology. Conclusion: Although the designed LAIV-RSV chimeric viruses were unable to neutralize the RS virus pulmonary replication, the LAIV-HA+G-RSV reduced RSV-induced lung pathology and can be considered a promising bivalent vaccine against influenza and RSV infections and warrants its further development.

scholarly journals Insertion of the Two Cleavage Sites of the Respiratory Syncytial Virus Fusion Protein in Sendai Virus Fusion Protein Leads to Enhanced Cell-Cell Fusion and a Decreased Dependency on the HN Attachment Protein for Activity

2008 ◽  
Vol 82 (12) ◽  
pp. 5986-5998 ◽  
Author(s):  
Joanna Rawling ◽  
Blanca García-Barreno ◽  
José A. Melero
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Cleavage Sites ◽  
Attachment Protein ◽  
F Protein ◽  
Syncytial Virus ◽  
Cell Cell

ABSTRACT Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (FRSV) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (FSeV), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both FRSV cleavage sites in FSeV resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric FSeV mutants containing both FRSV cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.

scholarly journals Identification of Temperature-Sensitive Mutations in the Phosphoprotein of Respiratory Syncytial Virus That Are Likely Involved in Its Interaction with the Nucleoprotein

2002 ◽  
Vol 76 (6) ◽  
pp. 2871-2880 ◽  
Author(s):  
Bin Lu ◽  
Robert Brazas ◽  
Chien-Hui Ma ◽  
Tina Kristoff ◽  
Xing Cheng ◽  
...  
Keyword(s):  
Amino Acids ◽  
Respiratory Syncytial Virus ◽  
Protein Function ◽  
Temperature Sensitive ◽  
P Gene ◽  
P Protein ◽  
Charged Residues ◽  
Cotton Rats ◽  
Syncytial Virus

ABSTRACT The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37°C in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39°C. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33°C, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37°C. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37°C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.

scholarly journals GS-5806 Inhibits Pre- to Postfusion Conformational Changes of the Respiratory Syncytial Virus Fusion Protein

2015 ◽  
Vol 59 (11) ◽  
pp. 7109-7112 ◽  
Author(s):  
Dharmaraj Samuel ◽  
Weimei Xing ◽  
Anita Niedziela-Majka ◽  
Jinny S. Wong ◽  
Magdeleine Hung ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Conformational Changes ◽  
Viral Entry ◽  
Viral Envelope ◽  
Host Cell Membrane ◽  
Virus Fusion ◽  
Molecule Inhibitor ◽  
Syncytial Virus

ABSTRACTGS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproducedin vitrousing a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity.

Sign in / Sign up

Export Citation Format

Share Document