title: Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2.
abstract: The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SB), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by E2-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each 11 mutants were identified. SB/R6, SB/R13, and SB-RL/R13 mutants contained a mutation at either E2 codon 96 or 159. SB-RL/R6 mutants contained changes at E2 codon 62, 96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E2c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice.
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290.
authors.forename: D F
authors.initials: DF
authors.lastname: Pence
authors.forename: N L
authors.initials: NL
authors.lastname: Davis
authors.forename: R E
authors.initials: RE
authors.lastname: Johnston
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000235:genetics*; D002460:Cell Line; D003062:Codon; D004797:Enzyme-Linked Immunosorbent Assay; D005814:Genes, Viral; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D009500:Neutralization Tests; D012845:Sindbis Virus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D013489:Suppression, Genetic; D014774:Virulence
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D003062:Codon; D006023:Glycoproteins