| Project/Area Number |
04557125
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用薬理学・医療系薬学
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| Research Institution | National Institute of Health |
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Principal Investigator |
MIYAMURA Tatsuo NIH,Dept.of Virology ll, Director, ウイルス2部, 部長 (90100099)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Yushiro St.Marianna Univ., Dept.of Inter.Med., Assistant Professor, 第2内科, 助教授 (00081688)
KATAYAMA Tohru National Tokyo Hospital, Director, 院長
SUZUKI Tetsuro NIH,Dept.of Virology ll, Researcher, ウイルス2部, 研究員 (00250184)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 1994: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1993: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1992: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Keywords | HCV / Serine protease / Anti-viral drug / Baculovirus / プロテアーゼ / 分子生物学 |
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| Research Abstract |
HCV contains a positive-stranded RNA genome with approximately 9,400 bases. It comprises a single, large open reading frame encoding a polyprotein of 3010-3033 amino acids, and it appears to be most closely related to flaviviruses and pestiviruses on the basis of similarities of its genomic structure and organization. Recent investigations revealed that four cleavages on the HCV precursor polyprotein are mediated by a viral serine-type proteinase lying within the amino-terminal half of the NS3 protein. This proteinase is thought to be a target for antiviral agents, and experimental systems of the enzyme activity will lead to testing therapeutic agents that might specifically inhibit HCV polyprotein processing. Our results are summarized as follows ;
(1) HCV NS3 serine proteinase was expressed in recombinant E.coli and baculovirus expression system, and the expressed protein exhibited specific proteolytic activity. (2) The enzyme was expressed in E.coli with His-Tag sequence at N-terminus and effectively purified with metal chelation resin. (3) High level expression of this enzyme was accomplished in baculovirus expression system by introducing rabies virus G protein signal sequence. (4) A human hepatoma cell line (HepG2) constitutively expressing proteins of HCV was established by transfection with HCV cDNA non-structural region. (5) A series of known proteinase inhibitors were tested by in vivo coexpression system and inhibition of the enzyme by TLCK was detected.
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