Phenotypic revertants of temperature-sensitive M protein mutants of vesicular stomatitis virus: sequence analysis and functional characterization

Abstract

Twenty-five spontaneous temperature-stable revertants of four different temperature-sensitive (ts) M protein mutants (complementation group III: tsG31, tsG33, ts023, and ts089) 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. Gopalakrishna 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.

Site-specific mutations in vectors that express antigenic and temperature-sensitive phenotypes of the M gene of vesicular stomatitis virus

“…Of considerable interest are early studies which indicate that various temperature-sensitive mutants of VSV in complementation group III contain M protein which exhibits a nonconditional loss in the phenotype for inhibition of VSV transcription (2, 29). Morita et al (14) have sequenced some of these mutants and many revertants by primer cDNA extension and found spontaneous nucleotide changes leading * Corresponding author.…”

Intragenic suppression of a deletion mutation of the nonstructural gene of an influenza A virus

“…Nucleotide sequencing revealed that there was a single-amino-acid difference between the NS gene segment of the 143-1 virus and the corresponding genes of the revertant viruses, a change from valine to alanine at amino acid 23 of the NS1 protein. Intragenic suppression of point mutations in ts mutants of influenza A viruses has been reported previously (18,26), as has intragenic suppression of i's and other mutations of animal viruses, including vesicular stomatitis virus, poliovirus, foot-and-mouth disease virus, vaccinia virus, and Sindbis virus (12,14,17). Intragenic suppression of a deletion mutation of an influenza virus has not been reported previously.…”

Membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus

“…This membrane-budding function is also shown to be restricted by a specific site mutation in the M-gene cDNA of a VSV temperature-sensitive mutant, in which phenylalanine is substituted for leucine at amino acid 111 of the M protein. This mutation previously had been shown to be responsible for restricted production of virions at a nonpermissive temperature (11), which can also be rescued by a coexpressed revertant (10).…”

“…We had previously also found that the M protein synthesized in cells infected with the M gene of temperature-sensitive mutant tsO23 failed to bind to plasma membrane at the restrictive temperature of 39ЊC but did so at the permissive temperature of 31ЊC (20). The M gene of tsO23 has three mutations leading to amino acid substitutions at residues 21, 111, and 227; the temperature-sensitivity restriction phenotype has been shown to be due to a Leu3Phe substitution at amino acid 111 (11). Plasmid expression of M protein with leucine at residue 111 will complement tsO23 infection by enhancing the release of virions at the restrictive temperature, but the presence of phenylalanine or other amino acids at residue 111 results in a failure to complement tsO23 infection (10).…”

“…We had also created deletion mutants of the M gene by restriction enzyme cleavage or by PCR which gave rise to truncated proteins in which the first 50 amino acids were deleted or amino acids 75 to 229 were deleted, as well as a double-deletion mutant missing amino acids 1 to 50 and 75 to 106 (20). In addition, the cDNA of the M gene of the temperature-sensitive mutant tsO23 had also been cloned in the same expression vector (10); the recombinant tsO23 M gene gave rise to a protein with amino acid substitutions at residues 21, 111, and 227 (11). Also available from earlier studies were mutant clones of the wt M gene which gave rise to proteins with single-amino-acid substitutions at residues 21, 111, and 227 identical to those of the tsO23 mutations, as well as revertants in the tsO23 M gene expressing proteins with individual revertants at amino acids 21, 111, and 227 (10).…”