A Pseudo-Revertant of a Sindbis Virus 6K Protein Mutant, Which Corrects for Aberrant Particle Formation, Contains Two New Mutations That Map to the Ectodomain of the E2 Glycoprotein

Abstract

Most site-directed mutations in the gene encoding the small, membrane-associated 6K protein of Sindbis virus interfere selectively with virus assembly and budding. Particles are released that are aberrant in structure, with a single membrane enclosing multiple nucleocapsids. A revertant for the mutation that inserted a serine for a cysteine at position 39 in the 6K protein was isolated and found to correct for the defective budding so that normal particles were formed. Genetic analysis of this revertant showed that two additional mutations, which were mapped to the ectodomain of the E2 virus glycoprotein, were present in addition to the original 6K substitution. The phenotype of the revertant differed from the wild-type strain and the original mutation with regard to plaque size, thermostability, and growth in neuronal cells. Five new virus genetic constructs were prepared by insertion of these mutations into the wild-type virus. Phenotypes of these constructs confirmed that the mutations in the E2 ectodomain were responsible for both correcting the original defect in budding as well as imparting changes 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 and bending during the budding process.