Development of anti-HCV drugs that inhibit HCV serine protease

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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.