Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides.
We have undertaken by biochemical and immunological experiments to locate the region of the matrix (M1) protein responsible for down-regulating endogenous transcription of A/WSN/33 influenza virus. A more refined map of the antigenic determinants of the M1 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 M1 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 M1 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 M1 protein. Of all the structural proteins of influenza virus, only NP and M1 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 M1 protein calculated by free-energy transfer suggests that the two hydrophilic transcription-inhibition RNA-binding domains are brought into close proximity by an alpha-helix-forming intervening hydrophobic domain.
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Department of Microbiology, University of Virginia Medical School, Charlottesville 22908.
Baylor
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Wagner
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002352:Carrier Proteins / Q000032:analysis*; D000939:Epitopes / Q000032:analysis; D007130:Immunoglobulin Idiotypes / Q000276:immunology*; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins; D011817:Rabbits; D014158:Transcription, Genetic; D014763:Viral Matrix Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D007130:Immunoglobulin Idiotypes; C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; D010446:Peptide Fragments; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014763:Viral Matrix Proteins