Development of anti-HCV drugs that inhibit HCV serine protease

HCV含有约9，400个碱基的正链RNA基因组。它包含编码3010-3033个氨基酸的多蛋白的单个大开放阅读框，并且基于其基因组结构和组织的相似性，它似乎与黄病毒和瘟病毒最密切相关。最近的研究表明，HCV前体多聚蛋白上的四个切割是由位于NS 3蛋白氨基末端一半内的病毒丝氨酸型蛋白酶介导的。这种蛋白酶被认为是抗病毒药物的靶标，并且酶活性的实验系统将导致测试可能特异性抑制HCV多蛋白加工的治疗剂。结果表明：（1）HCV NS 3丝氨酸蛋白酶在大肠杆菌和杆状病毒中得到了表达，表达产物具有特异性蛋白水解活性。(2)该酶在大肠杆菌中表达，N-末端带有His-标签序列，并用金属螯合树脂有效纯化。(3)通过引入狂犬病毒G蛋白信号肽序列，在杆状病毒表达系统中实现了该酶的高效表达。(4)用HCV cDNA非结构区转染人肝癌细胞株HepG 2，建立了组成型表达HCV蛋白的细胞系。(5)利用体内共表达系统检测了一系列已知的蛋白酶抑制剂，并检测了TLCK对该酶的抑制作用。

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.