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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >THE HEPATITIS C VIRUS NS3 SERINE PROTEINASE AND NS4A COFACTOR - ESTABLISHMENT OF A CELL-FREE TRANS-PROCESSING ASSAY
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THE HEPATITIS C VIRUS NS3 SERINE PROTEINASE AND NS4A COFACTOR - ESTABLISHMENT OF A CELL-FREE TRANS-PROCESSING ASSAY

机译：丙型肝炎病毒NS3丝氨酸蛋白酶和NS4A辅助因子-无细胞转运测定法的建立

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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. [References: 23]
机译：丙型肝炎病毒RNA基因组编码一个长的多蛋白，该蛋白经过蛋白水解加工成至少10种产物。这些裂解产物在该蛋白中的顺序为NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A- NS5B-COOH。除蛋白酶催化外，位于非结构蛋白NS3的N末端三分之一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。在4B / 5A位点而不是5A / 5B位点进行加工需要NS4A蛋白。这些裂解事件可能是病毒复制所必需的，这使丝氨酸蛋白酶成为有吸引力的抗病毒靶标。在这里，我们描述了一种体外测定方法，其中NS3-4A多聚蛋白NS3，丝氨酸蛋白酶域（NS3的N端181个残基），然后通过无细胞翻译产生NS4A辅因子，并测试其对放射性标记底物的反式加工能力。 NS4A-4B或截短的NS5A-5B多蛋白底物可被所有形式的蛋白酶反式切割，而NS4B-5A加工也需要NS4A。蛋白水解被取代突变取消，该取代突变先前显示出可灭活蛋白酶或阻断体内特定位点的裂解。此外，N末端序列分析确定体外裂解发生在真实的4A / 4B位点。在微粒体膜存在下的翻译增强了某些（但不是全部）蛋白酶-底物组合的加工过程，转加工是时间和温度依赖性的，并且通过在临界胶束浓度以上的各种去污剂处理消除了转加工。经过测试的抑制剂，只有高浓度（毫摩尔）的丝氨酸蛋白酶抑制剂甲苯磺酰氯甲基甲酮和4-（2-氨基乙基）苯磺酰氟可以使NS3蛋白酶失活。这种体外测定应有助于病毒丝氨酸蛋白酶的纯化和进一步表征以及分子鉴定选择性抑制其活性。 [参考：23]

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