A PSEUDO-REVERTANT OF A SINDBIS VIRUS 6K-PROTEIN MUTANT, WHICH CORRECTS FOR ABERRANT PARTICLE FORMATION, CONTAINS 2 NEW MUTATIONS THAT MAP TO THE ECTODOMAIN OF THE E2-GLYCOPROTEIN

Most site-directed mutations in the gene encoding the small, membrane- associated 6K protein of Sindbis virus interfere selectively with viru s assembly and budding, Particles are released that are aberrant in st ructure, with a single membrane enclosing multiple nucleocapsids. A re vertant for the mutation that inserted a serine for a cysteine at posi tion 39 in the 6K protein was isolated and found to correct for the de fective budding so that normal particles were formed. Genetic analysis of this revertant showed that two additional mutations, which were ma pped to the ectodomain of the E2 virus glycoprotein, were present in a ddition to the original 6K substitution. The phenotype of the revertan t differed from the wild-type strain and the original mutation with re gard to plaque size, thermostability, and growth in neuronal cells. Fi ve new virus genetic constructs were prepared by insertion of these mu tations into the wild-type virus. Phenotypes of these constructs confi rmed that the mutations in the E2 ectodomain were responsible for both correcting the original defect in budding as well as imparting change s in cell tropism, plaque size, and thermolability on the virus. These results indicate that 6K may play an indirect role in the packing of the virus spike glycoproteins, which allows for membrane deformation a nd bending during the budding process. (C) 1995 Academic Press, Inc.