Structure-based mutational analysis of the hepatitis C virus NS3 helicase
Structure-based mutational analysis of the hepatitis C virus NS3 helicase
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DOI：
10.1128/jvi.75.17.8289-8297.2001
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发表时间：
2001-09-01
影响因子：
5.4
通讯作者：
Chen, DS
中科院分区：
文献类型：
--
作者：
Tai, CL;Pan, WC;Chen, DS
丙型肝炎病毒（HCV）非结构蛋白3（NS3）的羧基端具有ATP依赖性RNA解旋酶活性。基于解旋酶结构域的保守序列基序和晶体结构，产生了17个HCV NS3解旋酶突变体。在体外检测突变蛋白的ATP水解、RNA结合和RNA解旋活性，以确定突变残基的功能作用。结果表明，步行者A基序中的Lys-210和步行者B基序中的Asp-290、Glu-291和His-293对ATP酶活性起决定性作用，基序III中的Thr-322和Thr-324和基序VI中的Arg-461对ATP酶活性有显著影响。当His-293和Gln-460之间的配对（称为看门人）被替换为Asp-293/His-460对时，这使得NS3解旋酶更像DEAD解旋酶亚组，ATP酶活性没有恢复。这表明，整个微环境周围的看门人，而不是残基本身，是重要的酶活性。Arg-461和Trp-501是RNA结合的重要残基，而瓦尔-432可能只起辅助作用。这些数据表明，RNA解旋酶活性可能被废除的ATP酶活性的损失或降低RNA结合活性。然而，低阈值水平的ATP酶活性被认为是足够的解旋酶活性。本研究结果为开发以NS3为靶点的抗HCV治疗药物提供了有价值的参考。
The carboxyl terminus of the hepatitis C virus (HCV)nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole micro environment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.