| Project/Area Number |
14572065
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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 |
Biological pharmacy
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| Research Institution | Kitasato University |
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Principal Investigator |
IWAMA Minako Kitasato University, School of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (10223421)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUMOTO Kiyohisa Kitasato University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (80092344)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Influenza virus / Endonuclease / mRNA / Cap structure / Transcription / RNA dependent RNA polymerase / Negative strand RNA genome / In vitro |
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| Research Abstract |
The Influenza virus (FluV) RNA polymerase exhibits a cap-dependent endonuclease activity, which cleaves host nuclear mRNAs to produce capped RNA fragments. The fragment functions as a primer to initiate viral mRNA synthesis. In this study, we analyzed the molecular mechanism of this FluV cap snatching reaction, and obtained the following results. (1) We developed an efficient in vitro endonuclease assay system using FluV-RNP. (2) We showed that methyl groups of cap structure were necessary for FluV type A endonuclease activity, but not for FluV type B, indicating that the recognition of cap structure by FluV type B is different from that by FluV type A. (3) Analyses of the capped RNA fragments, showed that they were 11 to 13 nucleotides in length, and that the 3'-end of the capped fragment is mainly A or G residue. In addition, we demonstrated that the 5'-end of the resulting 3'-RNA fragment formed after cleavage is mainly A residue. Thus, it was suggested that FluV endonuclease recognizes A or G residue at the position of 11 to 13 nucleotide from a cap structure and preferentially cleaves between A (or G) and A. (4) We attempted to develop a novel assay system using streptavidin-coated beads for anti-FluV endonuclease agents. After the substrate RNAs, which are radiolabeled in the cap structure and biotinylated at the 3' end, were incubated with FluV, RNAs were captured on streptavidin-coated beads. We showed that this assay system is useful for screening of anti-FluV agents.
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