Respiratory syncytial virus : complete nucleotide sequence of the fusion protein gene and identification of linear epitopes on the F1 subunit

Scopes, Geoffrey Eric (1989) Respiratory syncytial virus : complete nucleotide sequence of the fusion protein gene and identification of linear epitopes on the F1 subunit. University of Southampton, Doctoral Thesis.

Abstract

A cDNA library of Edinburgh strain (subtype A) Respiratory Syncytial (RS) virus infected cell mRNA was prepared and cloned into E.coli strain JM109. Initial screening yielded two RS virus specific clones, which DNA sequencing demonstrated to be the 1A (small hydrophobic protein) gene, and the Matrix protein (M) gene of RS virus Fusion protein (F) gene. The plasmid from this clone was designated pRSF1. Further screening produced a clone containing a full length copy of the RS virus. Complete sequencing of both strands of the insert indicated a sequence of 1906 nucleotides, with a single long open reading frame encoding an animo acid of 574 amino acids. Comparison with the previously published F-gene sequences demonstrated this sequence to have much closer homology with RS virus subtype A F-gene sequences than with the subtype B F-gene sequence. Sections of the F-gene sequence were inserted into expression vectors, designed to produce immunogenic polypeptides in E.coli. One of the resulting constructs, pRSF-SH, contained a 1kb insert consisting of virtually the entire F-gene, with the exception of the region encoding the cytoplasmic domain. The other construct, pRSF-PS, contained a 172bp fragment, including the coding region for the neutralizing epitope described by Trudel and colleagues (1987). The resulting constructs were either lethal in E.coli or failed to produce detectable F-gene products. Further analysis of the RS virus F-protein was carried out by multiple solid-phase chemical synthesis of peptides corresponding to the amino acid sequence derived from the nucleotide sequence of pRSF1. This demonstrated a region reactive with human hyperimmune sera at amino acids 478-492. Further analysis defined the peptide 483-FPSDEF-488 as the epitope, with amino acids P484 and E487 implicated as contact residues.

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