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 ts023(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 ts023 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 ts023 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.

Transcription inhibition site on the M protein of vesicular stomatitis virus located by marker rescue of mutant tsO23(III) with M-gene expression vectors

“…If the M protein being tested is devoid of transcription inhibition activity, RNA synthesis will increase by an amount commensurate with the increased concentrations of RNP template and its polymerase. As previously described (1), no transcription inhibition activity was exhibited by mutant M-protein present in leaky ts023 virions released from cells coinfected with the vaccinia virus recombinant vMts23 expressing Phe-111 M protein not capable of rescuing tsO23 (5). A similar pattern of increasing levels of RNA synthesis at increasing virus concentrations was exhibited by ts023 rescued by coinfection with revertant vMts23(R2) expressing M protein with Phe-111-*Leu amino acid substitution.…”

“…transcription inhibition by wt M protein (5,10). These and earlier results lead to the postulate that glycine at position 21 determines the three-dimensional structures at the N-terminal region of wt M protein that imparts its transcription inhibition activity as well as a major antigenic determinant (5, 10).…”

“…To identify more precisely the regions of the VSV M protein that serve as determinants for binding epitope 1specific MAb, for conversion to the temperature-sensitive phenotype, and for mediating transcription inhibition, we have cloned M-gene cDNAs of wt VSV-Indiana (Orsay strain) and its mutant tsO23 in procaryotic and eucaryotic expression vectors; we have also introduced by primer extension site-specific mutations in these clones and constructed wt-mutant chimeras (4,5). Expression in CV-1 cells of wt or mutant M genes cloned in plasmids under control of * Corresponding author.…”

“…vector vTF1-6,2. We used vaccinia virus recombinants with integrated plasmid pTF-OM79, pTF7-OM79(Glu21), pTF7-tsM23, or pTF7-tsM23(R2) rather than transfecting cells with the plasmids alone, because earlier studies have revealed yields of expressed M protein 4 to 15 times greater in cells coinfected with vaccinia virus M-gene recombinants (5). Each of these recombinants was created by integration into the vaccinia virus thymidine kinase locus of the respective plasmids with wt or mutant M genes flanked by the T7 polymerase promoter and terminator and then by transfection of vaccinia virus-infected CV-1 cells and selection of 5-bromodeoxyuridine-resistant recombinants (5).…”

Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids

“…Several lines of evidence suggest that the interaction between M protein and the nucleocapsid leading to transcription inhibition may not be the same as that involved in virus assembly. The temperature sensitivity of tsO23 maps to a Leu to Phe substitution at position 111 (5,12), a site that differs from that reported for transcription inhibition activity. Furthermore polylysine has transcription inhibition activity similar to that of the lysine-rich amino-terminal peptide of M protein (21), suggesting that many polycations may inhibit virus transcription.…”