MUTATIONS IN THE SINDBIS VIRUS CAPSID GENE CAN PARTIALLY SUPPRESS MUTATIONS IN THE CYTOPLASMIC DOMAIN OF THE VIRUS E2 GLYCOPROTEIN SPIKE

Assembly and budding of alphaviruses are postulated to occur by protei n-protein interactions between sites on the cytoplasmic domain of the transmembranal envelope E2 glycoprotein and on the surface of the nucl eocapsid protein subunits. Genetic data to support this model have bee n obtained by isolating revertants of two slow-growth mutants of Sindb is virus and analyzing the sequences of the genes encoding their struc tural proteins. The slow-growth phenotypes of the mutants were previou sly shown to result from site-directed mutations of 2 amino acids in t he sequence corresponding to the 33 amino acids at the carboxyl termin us of E2, which are localized to the cytoplasmic face of the plasma me mbrane. Putative revertants of these two mutants with faster growth ra tes were isolated by sequential passaging of virus grown on insect cel ls or chicken embryo fibroblasts. Sequence analysis of plaque-purified viruses that grew significantly better than the original mutant revea led that the original E2 mutation was present and that there were addi tional amino acid changes in the virus capsid. Two of the latter were introduced separately into the wild-type virus cDNA and into the genom es of the original mutants. The new strains of virus that contained bo th capsid and E2 mutations produced many more extracellular particles than those with the E2 mutations alone, indicating substantial suppres sion of the original E2 mutation. Both capsid mutations appear to be l ocalized near a hydrophobic pocket of the capsid, which is postulated to be the site for docking of hydrophobic amino acids of the E2 cytopl asmic domain. This genetic study provides strong support for the curre nt models of alphavirus assembly. (C) 1998 Academic Press.