A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop

Abstract

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.

Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

“…The conserved nature of the 13-residue sequence adjacent to and including 2 cysteines of the "cystine nooses" in G proteins of all human isolates of RSV, conservation of the cysteine residues in all RSV G proteins, and their presence as a "cystine noose" in both human and bovine isolates suggest a role for this subdomain in binding of RSV to susceptible cells. Furthermore, immunological studies have shown that this subdomain is a sub-group-specific immuno-dominant region of the G protein (Norrby et al, 1987;Akerlind-Stopner et al, 1990) that elicits neutralising antibodies due to natural infection (Norrby et al, 1987). Immunization of mice with this subdomain in synthetic peptide form (Trudel et al, 1991) or as part of expressed proteins (Olmstead et al, 1989;Sinard et al, 1995;Sullender et al, 1990) protected them from challenge with live RSV.…”

“…Immunization of mice with this subdomain in synthetic peptide form (Trudel et al, 1991) or as part of expressed proteins (Olmstead et al, 1989;Sinard et al, 1995;Sullender et al, 1990) protected them from challenge with live RSV. Immunological recognition of this subdomain is dependent upon intact disulfides (Akerlind-Stopner et al, 1990) and is subject to mutation in virus variants generated by propagation in the presence of neutralizing monoclonal antibodies (Rueda et al, 1994). Neutralizing antibodies that recognize this sub-domain also block binding of native G protein to RSV susceptible cells (Feldman & Hendry, 1994).…”

Evaluation of subgroup‐specific peptides of the G protein of respiratory syncytial virus for characterization of the immune response

“…Peptide 12, a single corresponding highly antigenic site in the G protein of subgroup A and subgroup B RSV, comprised of aa174-188 of both subgroups, reacts with homologous anti-G monoclonal antibodies in a site-specific manner. It appears to be the major reactive linear epitope of the G protein [Akerlind-Stopner et al, 1990;Norrby et al, 19871. Preliminary tests with paired sera from a small number of children undergoing naturally occurring RSV infection showed that very few of them devleoped rises in antibody during convalescence to peptide 12G [Akerlind-Stopner et al, 19901. To examine its value as a site-specific diagnostic antigen, we tested in this study a large number of serum pairs from children undergoing RSV infection.…”

Molecular characterization of Brazilian wild-type strains of bovine respiratory syncytial virus reveals genetic diversity and a putative new subgroup of the virus

“…Cys 173 -Cys 186 and Cys 176 -Cys 182 constitute the outer and inner disulfide bridges, respectively, while Asn 179 is involved in three hydrogen bonds that link the two helices of the Cys 176 -disulfide bridge via backbone oxygen with the Leu 183 amide in the second helix (Doreleijers et al 1996). In addition, although the cysteine noose was shown to be unnecessary for effective virus infection both in vitro and in vivo (Teng and Collins 2002), it was considered necessary for binding the virus to the cell (Akerlind-Stopner et al 1990, Valentova et al 2012. Therefore, it is likely that the lack of the three cysteines that were replaced by Leu, Tyr, and/or Arg and other residue mutations at positions 179 and 183 observed in the Brazilian BRSV strains in this study represent a loss of disulfide bridges and structural changes in the G protein.…”