Enhanced Mutability Associated with a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

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Abstract
Temperature-sensitive ( ts ) mutant ts D1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non- ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant ts D1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of ts D1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as ts D1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of ts D1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of ts D1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant ts D1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.