Phenotypic revertants of temperature-sensitive M protein mutants of vesicular stomatitis virus: sequence analysis and functional characterization.
Twenty-five spontaneous temperature-stable revertants of four different temperature-sensitive (ts) M protein mutants (complementation group III: tsG31, tsG33, tsO23, and tsO89) were sequenced and tested for their ability to inhibit vesicular stomatitis virus RNA polymerase activity in vitro. Consensus sequences of the coding region of each M protein gene were determined, using total viral RNA as template. Fifteen different sequences were found among the 25 revertants; 14 differed from their ts parent by a single amino acid (one nucleotide), and 1 differed by two amino acids (two nucleotides). Amino acids were altered in various positions between residues 64 and 215, representing over 60% of the polypeptide chain. Resequencing of the Glasgow and Orsay wild types and the four ts mutants confirmed previously published differences (Y. Gopalakrishana and J. Lenard, J. Virol., 56:655-659, 1985), and one or two additional differences were found in each. The relative charges of the revertant M proteins, as determined by nonequilibrium pH gradient electrophoresis, were consistent with the deduced sequences in every case. The ability of each revertant M protein to inhibit the RNA polymerase activity of nucleocapsids prepared from its parent ts mutant was also tested. Only 13 of the 25 revertants had M protein with high (wild type-like) polymerase-inhibiting activity, while 5 had low (ts-like) activity, and 7 had intermediate activity, demonstrating that this property is not an essential concomitant of the temperature-stable phenotype. It is concluded that the high reversion frequency observed for these mutants arises from a very high incidence of pseudoreversion, i.e., many different molecular changes can repair the ts phenotype.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005796:Genes; D005814:Genes, Viral; D005816:Genetic Complementation Test; D009154:Mutation; D010641:Phenotype; D013045:Species Specificity; D013696:Temperature; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014763:Viral Matrix Proteins; D014764:Viral Proteins / Q000235:genetics*
D014763:Viral Matrix Proteins; D014764:Viral Proteins