Differential roles of viral RNA and cRNA in functional modulation of the influenza virus RNA polymerase.
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.
Honda
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Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. ayhonda@lab.nig.ac.jp
Endo
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A
Mizumoto
K
K
Ishihama
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A
D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000502:physiology*; D008969:Molecular Sequence Data; D009975:Orthomyxoviridae / Q000201:enzymology*; D011061:Poly A / Q000378:metabolism; D018075:RNA, Complementary / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis / Q000502:physiology*
D018075:RNA, Complementary; D012367:RNA, Viral; D011061:Poly A; D012321:DNA-Directed RNA Polymerases