Antigenicity, function, and conformation of synthetic oligopeptides corresponding to amino-terminal sequences of wild-type and mutant matrix proteins of vesicular stomatitis virus

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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.

Section: Discussionmentioning

confidence: 81%

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confidence: 99%

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

Luo

Snyder

et al. 1988

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“…The critical role of the M1 protein in RNP-membrane assembly and budding to form progeny virions is becoming more apparent. The M protein of VSV has a similar size and similar functions but, unlike the influenza virus M, protein, the VSV M protein is basic, does not have membrane-intercalating hydrophobic sequences of amino acids, and has an N-terminal RNP-binding site that down-regulates transcription and a membranebinding site positioned toward the C terminus (17,20).…”

Section: Discussionmentioning

confidence: 99%

Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides

Baylor

Wagner

1989

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100

We have undertaken by biochemical and immunological experiments to locate the region of the matrix (M,) protein responsible for down-regulating endogenous transcription of A/WSNI33 influenza virus. A more refined map of the antigenic determinants of the M, protein was obtained by binding of epitope-specific monoclonal antibodies (MAbs) to chemically cleaved fragments. Epitope 2-specific MAb 289/4 and MAb 7E5 reverse transcription inhibition by M, protein and react with a 4-kilodalton cyanogen bromide fragment extending from amino acid Gly-129 to Gln-164. Anti-idiotype serum immunoglobulin G prepared in rabbits immunized with MAb 289/4 or MAb 7E5 mimicked the action of M, protein by inhibiting transcription in vitro of influenza virus ribonucleoprotein cores. This transcription-inhibition activity of anti-MAb 7E5 immunoglobulin G and anti-MAb 289/4 immunoglobulin G could be reversed by MAb 7E5 and MAb 289/4 or could be removed by MAb 7E5-Sepharose affinity chromatography. Transcription of influenza virus ribonucleoprotein was inhibited by one of three synthetic oligopeptides, a nonodecapeptide SP3 with an amino acid sequence corresponding to Pro-90 through Thr-108 of the Ml protein. Of all the structural proteins of influenza virus, only NP and Ml showed strong affinity for binding viral RNA or other extraneous RNAs. The 4-kilodalton cyanogen bromide peptide (Gly-129 to Gln-164), exhibited marked affinity for viral RNA, the binding of which was blocked by epitope 2-specific MAb 7E5 but not by MAbs directed to three other epitopes. Viral RNA also bound strongly to the nonodecapeptide SP3 and rather less well to anti-idiotype anti-MAb 7E5; these latter viral RNA-binding reactions were only slightly blocked by preincubation of anti-MAb 7E5 or SP3 with MAb 7E5. These experiments suggest the presence of at least two RNA-binding sites, which also serve as transcription-inhibition sites, centered around amino acid sequences 80 through 109 (epitope 4?) and 129 through 164 (epitope 2) of the 252 amino acid M1 protein of A/WSN/33 influenza virus. A hydropathy plot of the Ml protein calculated by free-energy transfer suggests that the two hydrophilic transcription-inhibition RNA-binding domains are brought into close proximity by an a-helix-forming intervening hydrophobic domain.

“…The M protein is quite basic and has a pl of -9.1 (6), largely because of a plethora of lysines and arginines at the amino terminus (23). The positively charged amino terminus of the M protein appears to provide the site for binding of M protein to RNP cores (10,24).…”

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confidence: 99%

Membrane-binding domains and cytopathogenesis of the matrix protein of vesicular stomatitis virus

Sun

Suryanarayana

et al. 1994

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The membrane-binding affinity of the matrix (M) protein of vesicular stomatitis virus (VSV) was examined by comparing the cellular distribution of wild-type (wt) virus M protein with that of temperature-sensitive (ts) and deletion mutants probed by indirect fluorescent-antibody staining and fractionation of infected or plasmid-transfected CVI cells. The M-gene mutant tsO23 caused cytopathic rounding of cells infected at permissive temperature but not of cells at the nonpermissive temperature; wt VSV also causes rounding, which prohibits study of M protein distribution by fluorescent-antibody staining. Little or no M protein can be detected in the plasma membrane of cells infected with tsO23 at the nonpermissive temperature, whereas-20% of the M protein colocalized with the membrane fraction of cells infected with tsO23 at the permissive temperature. Cells transfected with a plasmid expressing intact 229-amino-acid wt M protein (M1-229) exhibited cytopathic cell rounding and actin filament dissolution, whereas cells retained normal polygonal morphology and actin filaments when transfected with plasmids expressing M proteins truncated to the first 74 N-terminal amino acids (M1-74) or deleted of the first 50 amino acids (M51-229) or amino acids 1 to 50 and 75 to 106 (M51-74/107-229). Truncated proteins M1-74 and M51-229 were readily detectable in the plasma membrane and cytosol of transfected cells as determined by both fluorescent-antibody staining and cell fractionation, as was the plasmid-expressed intact wt M protein. However, the expressed doubly deleted protein M51-74/107-229 could not be detected in plasma membrane by fluorescent-antibody staining or by cell fractionation, suggesting the presence of two membrane-binding sites spanning the region of amino acids 1 to 50 and amino acids 75 to 106 of the VSV M protein. These in vivo data were confirmed by an in vitro binding assay in which intact M protein and its deletion mutants were reconstituted in highor low-ionic-strength buffers with synthetic membranes in the form of sonicated unilammelar vesicles. The results of these experiments appear to confirm the presence of two membrane-binding sites on the VSV M protein, one binding peripherally by electrostatic forces at the highly charged NH2 terminus and the other stably binding membrane integration of hydrophobic amino acids and located by a hydropathy plot between amino acids 88 and 119.