All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding
All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding
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DOI：
10.1128/jvi.00616-10
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发表时间：
2010-09-15
影响因子：
5.4
通讯作者：
Harris, Mark
中科院分区：
文献类型：
--
作者：
Foster, Toshana L.;Belyaeva, Tamara;Harris, Mark
丙型肝炎病毒（HCV）非结构蛋白NS5A对病毒基因组复制至关重要，并被认为与RNA依赖性RNA聚合酶NS5B和病毒RNA直接相互作用。NS5A由三个结构域组成，它们在病毒复制和组装中的作用尚未确定。为了确定介导与RNA（特别是HCV 3'非翻译区（UTR）正链RNA）相互作用的区域，克隆不同结构域组合的构建体，细菌表达，并纯化至均一。使用过滤器结合和凝胶电泳迁移率变动测定来探测这些纯化的蛋白质中的每一种与3'UTR RNA相互作用的能力，揭示它们的RNA结合效率和亲和力的差异。鉴定了NS5A的结构域I和II与3'UTR RNA之间的特异性相互作用，表明这些是NS5A的RNA结合结构域。结构域III显示低的体外RNA结合能力。滤膜结合和竞争分析鉴定了NS5A和NS5B之间在它们对3'UTR的限定区域的特异性方面的差异。与NS5B相反，NS5A对多聚嘧啶段的偏好突出了NS5A对3'UTR RNA识别的一个方面，这可能在控制或增强HCV基因组复制中起作用。
The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3' untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3' UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3' UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3' UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3' UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.