Purification and Characterization of the Respiratory Syncytial Virus Fusion Protein

Abstract

SUMMARYThe fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody. Under various conditions the protein was recovered as a 145K dimer or a 70K monomer. The 70K monomer was composed of disulphide-linked fragments of 48K and 23K. Polyclonal rabbit serum produced to the dimerized fusion protein neutralized virus but did not inhibit fusion, while rabbit serum to the 2-mercaptoethanol-treated dimerized protein neutralized virus and inhibited fusion of infected cells. Only the latter serum strongly recognized the 23K fragment when studied by Western blot analysis.

Intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage

“…6). The non-reduced F proteins synthesized by RSV or vF317 migrated on SDS-polyacrylamide gels both as F protein monomers (F), which include both F0 and mature F that has been cleaved to form F1 and F2, and F protein oligomers, which confirmed the previous observation that F protein forms oligomers (Walsh et al, 1985;Arumugham et al, 1989a). In addition, F337 and F373 produced an amount of oligomeric F protein consistent with the reduced amount of cleaved product observed for these proteins in Fig.…”

“…Previous work has indicated that the mature RSV F protein exists primarily as an oligomer, a non-covalently linked dimer consisting of two mature F protein molecules (Walsh et al, 1985;Arumugham et al, 1989a). To determine whether the wt and mutant F proteins were able to assemble into oligomers, non-reduced immuneprecipitated samples from the previous pulse-chase experiment (shown in Fig.…”

“…A membrane anchor region consisting of 26 hydrophobic amino acid residues is found near the C terminus of F1, indicating that the F protein has a structural orientation typical of class I membrane glycoproteins. Post-translational modifications of F protein in addition to cleavage of the signal peptide and cleavage of Fo to form the F1 and Fz subunits include the formation of oligomers (Walsh et al, 1985;Arumugham et al, 1989a), the covalent attachment of palmitate to cysteine residue 550, located in the membrane anchor region (Arumugham et al, 1989b), and the addition of N-linked carbohydrates (Gruber & Levine, 1985a;Peeples & Levine, 1979). There are four potential N-linked glycosylation sites on the F2 subunit and one on the F1 subunit Elango et al, 1985).…”

Lactoferrin and surfactant protein A exhibit distinct binding specificity to F protein and differently modulate respiratory syncytial virus infection

“…3A, lane a) and the fragments of F 1 and F 2 were recovered under reducing conditions at molecular masses of 48 kDa and 23 kDa, respectively (Fig. 3A, lane b), which were consistent with the previous study [17].…”

Passively Acquired Antibodies Suppress Humoral But Not Cell-Mediated Immunity in Mice Immunized with Live Attenuated Respiratory Syncytial Virus Vaccines

“…Serology. Serum IgG Abs binding to RSV F surface glycoprotein were quantitated in an ELISA using F glycoprotein that had been immunoaffinity-purified from RSV subgroup A (Long strain)-infected cell lysates as described (19,27). The neutralizing Ab titer of serum samples was determined by a complement-enhanced 60% plaque reduction assay in HEp-2 cells (28); plaques were visualized using the RSV-specific immunohistological staining procedure (26).…”