| Project/Area Number |
15570150
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Nippon Institute for Biological Science |
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Principal Investigator |
HONDA Ayae Nippon Institue for Biological Science, Research Division, Senior Scientist, 研究部, 研究員 (80343747)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIHAMA Akira Nippon Institue for Biological Science, Research Division, Principal Investigator, 研究部, 主任研究員 (80019869)
IWATA Akira Nippon Institue for Biological Science, Research Division, Principal Investigator, 研究所, 主任研究員 (70193745)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | vRNA / cRNA / RNA-dependent RNA polymerase / endonuclease / effector RNA / 複製 / インフルエンザウイルス / RNAポリメラーゼ / エンドヌクレアーゼ / RNAゲノム / 組換えバキュロウイルス / RNAエフェクター / 酵素活性制御 / 転写活性 / RNA構造 / RNAエンドヌクレアーゼ |
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| Research Abstract |
The genome of influenza virus consists of eight segments of negative-strand RNA. Both transcription and replication of the viral genome is catalyzed by the influenza virus RNA-dependent RNA polymerase, which is composed of three viral protein subunits, PB1, PB2 and PA. The aim of this research was to understand how the functions of influenza virus RNA-dependent RNA polymerase were regulated. We succeeded the purification of enzymatically active RNA-dependent RNA polymerase from insect cells co-infected with three species of the recombinant baculovirus, each expressing one of three subunits. We analyzed the activity and specificity of the purified RNA polymerase, and got several novel findings as follows :
1)The purified RNA polymerase gained the enzyme activity, as measured by endonucleolytic cleavage of capped RNA, only when viral RNA (vRNA) was added. We then proposed a model that vRNA has an "effector" role in controlling the enzyme activity.
2)This finding encouraged us to identify t
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he essential RNA element needed for the effector function. Various recombinant plasmids were constructed, which express the chimeric promoters between vRNA and cRNA. After analysis of the effector activity for these artificial promoters, we found that the bulge structure in the promoter region was necessary for activation of the RNA polymerase.
3)The RNA polymerase required primers, ApG or capped poly(A), for vRNA-dependent synthesis of RNA as in the case of viral RNP-associated RNA polymerase. This indicates that the RNA polymerase carries the specificity of transcriptase. However, the activity of cRNA-dependent unprimed RNA synthesis was very weak, in agreement with our finding that a host factor(s) is involved in the functional conversion of trascriptase to replicase. Candidates of the putative host factor have been identified by yeast two-hybrid screening.
4)Primer Binding site on PB1 was identified using radioactive primer (ApG), which was synthesized using ATP analogue and radioactive GTP. After cross-linking of the radioactive primer, we identified the primer-binding site after digestion of PB1 protein by protease. The primer-binding region was located close to the catalytic site of RNA synthesis on the PB1 subunit Less
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