期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 276, 期 33, 页码 31179-31185出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M102856200
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The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multifunctional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA binding, capped RNA cleavage, viral model RNA binding, model RNA-directed RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vRNA but not of cRNA, indicating that the vRNA functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates.
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