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

Abstract

Full-length cDNA copies of mRNAs coding for the matrix (M) proteins of vesicular stomatitis virus and its mutant tsO23(III) were cloned in pBSM13- (BlueScribe). The authenticity of these clones was demonstrated by restriction enzyme mapping, DNA sequencing, and in vitro transcription and translation to identify the two M proteins by Western immunoblotting with epitope-specific monoclonal antibodies. Site-directed mutants were constructed by primer extension of synthetic oligodeoxynucleotides with one or two nucleotide changes to alter the glycine at amino acid 21 of the wild-type (wt) M gene to glutamic acid, alanine, or proline. Similarly, a revertant was created in the M gene of mutant tsO23 by a Glu-21----Gly substitution. A series of wt- and mutant-M-gene chimeras was also constructed to create mutant and revertant clones with Leu----Phe and His----Tyr alterations at amino acids 111 and 227, respectively. We then moved the wt and tsO23 M genes and their site-specific mutants and chimeras cloned in pBSM13- into the eucaryotic expression vector pTF7 directed by the T7 bacteriophage RNA polymerase of the vaccinia virus recombinant vTF1-6,2. Western blot analysis of the M proteins transiently expressed in CV-1 cells by plasmids carrying M genes altered at amino acid 21 revealed that the critical antigenic determinant (epitope 1) is expressed only by the Gly-21 M protein and not by Glu-21, Ala-21, or Pro-21 M proteins. Of particular interest is an apparent conformational change, evidenced by slightly but significantly retarded electrophoretic migration, in plasmid-expressed M proteins with amino acids substituted for glycine at position 21. The glutamic acid at position 21 of tsO23 is not responsible for its temperature-sensitive phenotype, because a tsO23 revertant plasmid with glycine substituted at position 21 fails to rescue tsO23 virus in cells infected at the restrictive temperature; conversely, plasmids expressing wt M protein with substitutions of glutamic acid, alanine, or proline at position 21 are just as effective in marker rescue of tsO23 as is the Gly-21 wt M protein. Marker rescue experiments with wt- and mutant-M-gene chimeras support the hypothesis of K. Morita, R. Vanderoef, and J. Lenard (J. Virol. 61:256-263, 1987) that the temperature-sensitive phenotype of tsO23 is due to a phenylalanine substituted for leucine at amino acid 111, rather than the His-227----Tyr substitution or the Gly-21----Glu substitution, which independently accounts for the loss of epitope 1 in the mutant M protein of tsO23.(ABSTRACT TRUNCATED AT 400 WORDS)