Lin C, Rice C M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110-1093, USA.
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7622-6. doi: 10.1073/pnas.92.17.7622.
The hepatitis C virus RNA genome encodes a long polyprotein that is proteolytically processed into at least 10 products. The order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase domain located in the N-terminal one-third of nonstructural protein NS3 mediates cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites). In addition to the proteinase catalytic domain, the NS4A protein is required for processing at the 4B/5A site but not at the 5A/5B site. These cleavage events are likely to be essential for virus replication, making the serine proteinase an attractive antiviral target. Here we describe an in vitro assay where the NS3-4A polyprotein, NS3, the serine proteinase domain (the N-terminal 181 residues of NS3), and the NS4A cofactor were produced by cell-free translation and tested for trans-processing of radiolabeled substrates. Polyprotein substrates, NS4A-4B or truncated NS5A-5B, were cleaved in trans by all forms of the proteinase, whereas NS4A was also required for NS4B-5A processing. Proteolysis was abolished by substitution mutations previously shown to inactivate the proteinase or block cleavage at specific sites in vivo. Furthermore, N-terminal sequence analysis established that cleavage in vitro occurred at the authentic 4A/4B site. Translation in the presence of microsomal membranes enhanced processing for some, but not all, proteinase-substrate combinations. Trans-processing was both time and temperature dependent and was eliminated by treatment with a variety of detergents above their critical micelle concentrations. Among many common proteinase inhibitors tested, only high (millimolar) concentrations of serine proteinase inhibitors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)benzenesulfonyl fluoride inactivated the NS3 proteinase. This in vitro assay should facilitate purification and further characterization of the viral serine proteinase and identification of molecules which selectively inhibit its activity.
丙型肝炎病毒RNA基因组编码一种长多聚蛋白，该多聚蛋白经蛋白酶水解加工成至少10种产物。这些切割产物在多聚蛋白中的顺序为NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH。位于非结构蛋白NS3 N端三分之一区域的丝氨酸蛋白酶结构域介导在四个下游位点（3/4A、4A/4B、4B/5A和5A/5B位点）的切割。除蛋白酶催化结构域外，NS4A蛋白是4B/5A位点加工所必需的，但不是5A/5B位点加工所必需的。这些切割事件可能对病毒复制至关重要，这使得丝氨酸蛋白酶成为一个有吸引力的抗病毒靶点。在此，我们描述了一种体外检测方法，其中NS3-4A多聚蛋白、NS3、丝氨酸蛋白酶结构域（NS3的N端181个残基）和NS4A辅助因子通过无细胞翻译产生，并测试其对放射性标记底物的反式加工。多聚蛋白底物NS4A-4B或截短的NS5A-5B被所有形式的蛋白酶反式切割，而NS4B-5A加工也需要NS4A。先前显示可使蛋白酶失活或在体内阻断特定位点切割的取代突变消除了蛋白水解作用。此外，N端序列分析确定体外切割发生在真实的4A/4B位点。在微粒体膜存在下进行翻译增强了某些但不是所有蛋白酶-底物组合的加工。反式加工既依赖时间也依赖温度，并且通过用高于其临界胶束浓度的多种去污剂处理而消除。在测试的许多常见蛋白酶抑制剂中，只有高浓度（毫摩尔）的丝氨酸蛋白酶抑制剂甲苯磺酰赖氨酰氯甲基酮和4-（2-氨基乙基）苯磺酰氟可使NS3蛋白酶失活。这种体外检测方法应有助于病毒丝氨酸蛋白酶的纯化和进一步表征以及选择性抑制其活性的分子的鉴定。
