Quezada Elizabeth M, Kane Caroline M
Department of Molecular and Cell Biology, University of California, Berkeley CA 94720-3202, USA ; Stowers Research Institute 1000 East 50th Street Kansas City, MO 64110, USA.
Open Biochem J. 2013;7:11-4. doi: 10.2174/1874091X01307010011. Epub 2013 Jan 31.
The Hepatitis C Virus RNA dependent RNA polymerase, NS5B, is stimulated by the NS5A protein in vitro. To explore this stimulatory mechanism, we compared the activity of a mutant of NS5B containing a deletion of the β-loop region with that of the full length NS5B in response to NS5A. While the NS5A protein does stimulate full length NS5B, NS5A does not stimulate the NS5B deletion mutant during either replication initiation or elongation. This result suggests that the activation mechanism might involve a NS5A-mediated conformational change of the β-loop of NS5B. Such a conformational change would be predicted to prevent steric clash of the RNA template and newly synthesized RNA product. Consistent with this hypothesis, RNA binding is enhanced when the full length NS5B and NS5A are incubated with RNA, but RNA binding is unchanged with incubation of NS5A and the NS5B β-loop deletion mutant.
丙型肝炎病毒RNA依赖性RNA聚合酶NS5B在体外受到NS5A蛋白的刺激。为了探究这种刺激机制，我们比较了一个缺失β-环区域的NS5B突变体与全长NS5B在响应NS5A时的活性。虽然NS5A蛋白确实能刺激全长NS5B，但在复制起始或延伸过程中，NS5A都不会刺激NS5B缺失突变体。这一结果表明，激活机制可能涉及NS5A介导的NS5Bβ-环构象变化。预计这种构象变化会防止RNA模板与新合成的RNA产物发生空间冲突。与这一假设一致的是，当全长NS5B和NS5A与RNA一起孵育时，RNA结合增强，但NS5A与NS5Bβ-环缺失突变体孵育时，RNA结合没有变化。
