Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus.

Abstract

The genetic characterization of four previously reported mutants of human respiratory syncytial (RS) virus resistant to monoclonal antibody 63G is described. Sequences of the G protein genes were obtained from: (i) mRNA derived cDNA recombinants, (ii) direct mRNA sequencing and (iii) amplified vRNA derived cDNAs. The results obtained indicate that the original escape mutants, recovered from individual plaques, contained heterogeneous viral populations. This heterogeneity affected the number of adenosine residues present after nucleotides 588 or 623 of the G protein gene. Mutant viruses recovered after a second plaque purification step generated homogeneous sequences but contained single adenosine insertions or deletions at those two sites compared with the Long sequence. These genetic alterations introduced frameshift changes which are reflected in both the antigenic and structural properties of the mutant G proteins. The origin and importance of frameshift mutations in the RS virus G protein gene are discussed.

Oligomerization and post-translational processing of glycoprotein G of human respiratory syncytial virus: altered O-glycosylation in the presence of brefeldin A

“…The G protein also contains a substantial amount of O-linked sugars which, among non-segmented negative-strand viruses, have also been reported only in the filoviruses (Feldmann et al, 1991). The ectodomain contains more than 70 serine residues as potential acceptor sites; their number and distribution suggests that the G protein might contain numerous side-chains scattered on either side of the conserved domain, and this model has some support from studies of truncated G proteins (Olmsted et al, 1989;Garcia-Barreno et al, 1990). The unusual amino acid composition and glycosylation of the ectodomain suggests that it might have an extended nonglobular structure (Jentoft, 1990).…”

The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity

“…It was grown in Hep-2 cells and purified from culture supernatants as previously described (Garcia-Barreno et aL, 1988). The selection of the mutant viruses (R63/2/4/1, R63/4/10]5 and R63/8/7/2) resistant to MAb 63G has been described (Garcia-Barreno et al, 1990).…”

Location of the Epitope Recognized by Monoclonal Antibody 63G on the Primary Structure of Human Respiratory Syncytial Virus G Glycoprotein and the Ability of Synthetic Peptides Containing this Epitope to Induce Neutralizing Antibodies

“…The Long strain (subgroup A) of human RSV was used as the prototype throughout. The isolation and characterization of mutants R63/1/2/3, R63/2/4/1 and R63/2/4/8, which escape neutralization by MAb 63G, have been reported (Garcia-Barreno et al, 1990). The viruses were grown in monolayers of actively growing HEp-2 cells and purified from the culture supernatant as described (Garcia-Barreno et al, 1988).…”

“…Thus, the R63/2/4/1 sequence predicts a G protein with seven amino acid changes (205 to 211), but of wild-type length. This protein loses only the binding site for MAb 63G (Garcia-Barreno et al, 1990).…”

“…In addition, the G protein also shows extensive antigenic (Anderson et al, 1985;Akerlind et al, 1988;Garcia-Barreno et al, 1989) and genetic (Cane et al, 1991 ;Sullender et al, 1991) diversity among viruses of the same antigenic subgroup. The analysis of escape mutants selected with anti-G protein monoclonal antibodies (MAbs) indicates that antigenic variants of the G protein may be generated by one of three mechanisms: (i) point mutations, (ii) premature stop codons (Rueda et al, 1991) or (iii) frameshift mutations (Garcia-Barreno et al, 1990).…”