| Project/Area Number |
14370054
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | KANAZAWA UNIVERSITY |
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Principal Investigator |
MURAKAMI Seishi Cancer Research Institute, Dept.of Mol.Oncology, Professor, がん研究所, 教授 (90019878)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Naoyuki Cancer Research Institute, Dept.of Mol.Oncology, Research Associate, がん研究所, 助手 (50253456)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥11,900,000 (Direct Cost: ¥11,900,000)
Fiscal Year 2004: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2002: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | HCV / NS5B / NS5A / RNA replicon / Nucleolin / RNA dependent RNA polymerase (RdRP) / library of clustered alanine substitution mutant / HCV NS5B / アラニンスキャニング / 鋳型 / プライマー結合 / HCV複製 / オリゴマー |
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| Research Abstract |
Eradication of HCV persistent infection may prevent or delay incidence of hepatocellular carcinoma. HCV NS5B is RdRP that is a strong target to eradicate HCV replication. In hope to find novel strategies, we had identified 5 residues of NS5B critical for RdRP activity by introducing clustered alanine substitution (Cm) and point alanine substitution. This grant aims to get further insight of structure and function of RdRP enzyme, to characterize NS5B-interacting partners, NS5A and a host factor nucleolin, and finally to evaluate these interactions and mutations of NS5B on HCV RNA replication using a HCV RNA replicon system. Followings are the results we obtained.
1)Oligomer formation of NS5B was observed and two residues were defined by scanning the Cm and Pm libraries to be critical for oligomer formation. Interestingly the two residues are among the residues indispensable for RdRP activity, strongly suggesting that oligomeric interaction might be prerequisite for RdRP activity. 2)Speci
… More
fic interaction between NS5B and NS5A was found. The critical regions of the two factors for the interaction were mapped, 3)We found NS5B truncated at C-terminal was enriched in nucleoli. Nucleolin was defined to the interacting factor with NS5B. The NS5B-binding fragments did not affected much RdRP activity, although the interaction affected the subcellular localization of NS5B. 4)We evaluated whether the critical 5 residues for RdRP in vitro are indispensable for HCV replication using MlLE HCV RNA replicon. Actually all the residues are absolutely required for HCV replication. 5)Using HCV replicon harboring mutations of NS5A that are defective in NS5B-binding, we evaluated the role of the NS5A-NS5B interaction in HCV replication. Cm mutants and some Pm mutants within the regions necessary for NS5B-binding could not support HCV replication, strongly suggesting the importance of the inter action. 6)We evaluated the interaction of nucleolin and NS5B in the HCV RNA replicon system. Cm mutant of NS5B defective in nucleolin-binding could not support efficient HCV replication. Transient knock-down of nucleolin with siRNA greatly reduced the G418-forming foci, strongly suggesting that the interaction of nucleolin and NS5B is critical for HCV replication. Less
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