Directed Mutagenesis of a Sindbis Virus Pathogenesis Site

The E2 glycoprotein gene of Sindbis virus contains three neutralizing antigenic sites, and examination of monoclonal antibody (MAb) escape mutants of one of these, the E2c site, has suggested an association of the E2c domain with determinants of virulence in neonatal mice. Codons associated with the E2c site, E2 62, 96, and 159, were subjected to site-directed mutagenesis generating a library of 25 viable virus mutants containing different amino acids at these loci. The mutants were assessed for their ability to bind E2a, E2b, and E2c MAbs in an enzyme-linked immunosorbent assay, to induce fatal disease in neonatal mice following subcutaneous or intracranial inoculation and to compete with wild-type virus for binding to unfractionated neonatal mouse brain cells. None of the substitutions affected binding of E2a or E2b MAbs, and decreased binding to E2c MAbs was correlated with decreased virulence in neonatal mice. However, correlation of decreased virulence and binding to E2c MAbs with decreased competition for wild-type virus binding to mouse brain cells was not observed. The effect of mutation on E2c binding and virulence varied widely depending on the locus and the specific substitution, suggesting strategies for the molecular design of live virus vaccines which select both locus and codon to maximize attenuation and minimize the potential for reversion to virulence.