Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge (original) (raw)
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Journal of Virology, 2008
Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.
Journal of General Virology, 1996
The attachment protein G of human respiratory syncytial (RS) virus is a type II transmembrane glycoprotein. A secreted form of the G protein is also produced. To examine the two distinct hydrophobic regions in the Nterminal 63 amino acids of G protein for their role(s) in membrane insertion and anchoring, transport to the cell surface, and secretion, G proteins that contained point mutations or deletions were synthesized by cell-free transcription-translation and in cells by expression from recombinant vaccinia virus vectors. A mutant protein lacking the entire major hydrophobic region (amino acids 38-63) was not glycosylated, not expressed on the cell surface, and not secreted, because it was not inserted into membranes. In contrast, deletion of the minor hydrophobic region (amino acids 23-31) had no detectable effect on membrane insertion or anchoring. These data provided direct evidence that amino acids 38-63 were necessary for membrane insertion and contained the signal/anchor domain of RS virus G protein. Mutant proteins that lacked either the Nterminal or the C-terminal half of this 26 residue hydrophobic region were inserted into membranes and processed to maturity, showing that either half of this region was sufficient for membrane insertion. However, these two mutant proteins were secreted more abundantly than wild-type G protein. We propose that their truncated hydrophobic domains interacted with membranes in a way that mimicked the N-terminal signal sequence of naturally secreted proteins, allowing proteolytic cleavage of the mutant proteins.
Virology, 2002
Cell surface glycosaminoglycans (GAGs) are responsible for the majority of respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the viral attachment protein, but more recently virus lacking the G protein was shown to be infectious in cell culture. We have compared the GAG dependence of a recombinant, green fluorescent protein-expressing virus containing the F protein as its only viral glycoprotein (rgRSV-F) to isogenic complete virus containing all three viral glycoproteins (rgRSV-SGF). Attachment and infection by each virus was found to be largely dependent on cell surface heparan sulfate (HS) based on the finding that both activities were inhibited by preincubation of virus with soluble HS, by removal of HS from target cells by enzymatic treatment or mutation, or by pretreatment of the target cells with basic fibroblast growth factor (bFGF), which binds HS. These results, coupled with the previous finding that SH is not involved in virion binding (S. Techaarpornkul, N. Barretto, and M. Peeples, 2001, J. Virol. 75, 6825-6834), suggest that, in the context of the virion, both the G and F proteins bind to HS. Interestingly, both rgRSV-F and rgRSV-SGF retained significant binding activity and infectivity despite these treatments, suggesting an alternate productive attachment and infection pathway. This property of GAG independence was particularly apparent for rgRSV-F virions, which retained nearly half of its attachment and infection activities in most of these experiments. Comparison of the attachment and infection activities of rgRSV-SGF and rgRSV-F virions with a Chinese hamster ovary cell line and a derivative thereof that is defective in GAG synthesis indicated that approximately 50% of rgRSV-SGF attachment is due to G protein-GAG binding, 25% to F protein-GAG binding, and 25% to an independent pathway. This alternative pathway presumably is mediated by the sole remaining viral surface protein, F, although the formal possibility exists that some other virion-associated protein is involved.
Virus Research, 2019
Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F Nglycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F Nglycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches. Keywords Orthopneumovirus-fusion protein-N-glycosylation-recombinant virus recovery-antigenicity 1 Introduction The respiratory syncytial virus (RSV) is a major cause of infant morbidity and mortality related to lower respiratory tract disease. The disease burden in children younger than 5 years is estimated at 33.8 million infections annually from which 10% requires hospitalization (Nair et al., 2010). A vaccine to control the RSV disease burden remains elusive and treatment options are mainly supportive. Palivizumab, a humanized monoclonal antibody which targets a conserved epitope of the RSV fusion (F) protein, is able to reduce RSV-related hospitalizations when prophylactically administered. However, its use is restricted to high-risk children due to the high cost and the requirement of monthly intramuscular injections throughout the RSV season (Homaira et al., 2014). Re-infections are very common which is assumed to be the consequence of an incomplete and short-lived immunity upon natural infection. 2 Material and methods 2.1 Cells and virus The human epidermoid carcinoma larynx (HEp-2) and Vero cell lines were obtained from the ATCC. The cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% inactivated fetal bovine serum (iFBS). BSR T7/5 cells were a gift of K.K.
Canadian Journal of Microbiology, 1987
Competition experiments and biological assays with a panel of 15 monoclonal antibodies confirmed the presence of at least four antigenic sites on the fusion protein of human respiratory syncytial virus, three of which were involved in virus neutralization. One antigenic site, recognized by two strongly neutralizing antibodies, was conserved after reduction and denaturation and shown by immunoblotting to be localized on the F1 fragment of the fusion protein. Cleavage of this protein with staphylococcal protease V8 or papain produced a series of smaller peptides from 11 to 7 kilodaltons that retained this important neutralization determinant. Compared with the other neutralization sites, the epitope defined by monoclonal antibody 7C2 thus appears as the major neutralization epitope. Our peptide mapping results support the hypothesis that this major epitope is composed of a continuous sequence on the viral genome.
Respiratory syncytial virus envelope glycoprotein (G) has a novel structure
Nucleic Acids Research, 1985
ASTno acid sequence of human respiratory syncytiai virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino add microsequendng of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated J[n vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane Insertion at 41 residues from the N-term1nus. There was no N-terminal signal sequence and the hydrophHic N-term1nal 20 residues probably represent the cytoplasmic tall of the protein. The N-terminally oriented membrane Insertion was somewhat analogous to paramyxovims hemagglutinin-neuraminidase (HN) and Influenza neuraminidase (NA). The protein was moderately hydrophilic and rich 1n hydroxy-amino adds. It was both N-and O-glycosylated with the latter contributing significantly to the net molecular mass 90K.
Virology, 1996
Infection of different human epithelial cell lines with human respiratory syncytial virus (HRSV) revealed significant differences in the electrophoretic mobility of the viral attachment glycoprotein (G). Cell-type specific differences in G protein glycosylation were observed with certain lectins and sugar-specific reagents. Furthermore, substantial changes in the reactivity of the G glycoprotein with anti-G monoclonal antibodies were associated to the infected cell type. Strain-specific epitopes-present in a limited number of HRSV isolates of the same antigenic group-were particularly susceptible to celltype-specific modifications of the mature G protein. Some of these epitopes, which were either exposed in the unglycosylated precursor or reproduced with synthetic peptides, were nonetheless masked in the mature G protein expressed in certain cell lines. Antigenic and electrophoretic mobility changes of the G glycoprotein were reverted in extracts of HEp-2 cells infected with HRSV grown in other cell types, indicating that phenotypic traits rather than selection of variants were associated to the above stated changes. These results highlight the importance of cell-type-specific modifications for HRSV G glycoprotein antigenicity and raise questions about the actual antigenic structure of this molecule when HRSV replicates in the respiratory tract. ᭧
Journal of General Virology, 1992
Two antigenic sites recognized by neutralizing monoclonal antibodies (MAbs) directed against the fusion (F) glycoprotein of human respiratory syncytial virus were mapped on the primary structure of the protein by (i) the identification of amino acid substitutions selected in antibody-escape mutants and (ii) the reactivity of synthetic peptides with MAbs. The first site contained several overlapping epitopes which were located within the trypsin-resistant amino-terminal third of the large F1 subunit. Only one of these epitopes was faithfully reproduced by a short synthetic peptide; the others might require specific local conformations to react with MAbs. The second antigenic site was located in a trypsin-sensitive domain of the F1 subunit towards the carboxy-terminal end of the cysteine-rich region. One of these epitopes was reproduced by synthetic peptides. In addition, mutagenized F protein with a substitution of serine for arginine at position 429 did not bind MAbs to the second site. These results are discussed in terms of F protein structure and the mechanisms of virus neutralization.
Journal of Virology
The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or...