Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein.
Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.
Tokyo Institute of Technology, Yokohama, Japan.
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K
Watanabe
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Handa
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Mizumoto
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Nagata
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009980:Influenza A virus / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014763:Viral Matrix Proteins / Q000378:metabolism*
D017931:DNA Primers; D004791:Enzyme Inhibitors; C031540:M-protein, influenza virus; C488936:M1 protein, Influenza A virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases