Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions
Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions
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DOI：
10.1128/jvi.02446-09
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发表时间：
2010-06-01
影响因子：
5.4
通讯作者：
Kao, C. C.
中科院分区：
文献类型：
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作者：
Chinnaswamy, S.;Murali, A.;Kao, C. C.
丙型肝炎病毒（HCV）RNA依赖性RNA聚合酶（RdRp）已被提出与RNA合成相关的构象改变以及与细胞蛋白质相互作用。在体外，RdRp可以从单链RNA的末端从头开始或延伸引发的RNA模板。NS 5 B中Delta 1环和拇指结构域之间的相互作用是从头起始所需的，尽管尚不清楚这些相互作用是在NS 5 B单体内还是高阶NS 5 B寡聚复合物的一部分。这项工作旨在解决聚合酶构象和/或寡聚化如何影响从头开始。我们已经表明，增加酶浓度增加从头启动基因型1b和2a RdRps，而引物延伸反应不受影响或抑制在类似的条件下。HCV聚合酶的起始缺陷突变体可以增加野生型（WT）聚合酶的从头起始。GTP也被发现刺激从头开始。我们的研究结果支持一个模型，其中从头启动能力构象的RdRp的刺激，通过寡聚体之间的接触个别亚基。使用电子显微镜和单分子重建，我们试图可视化的低分辨率构象的二聚体从头启动能力的HCV RdRp。
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Delta 1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.