Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation
Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation
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DOI：
10.1074/jbc.m803094200
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发表时间：
2008-12-05
影响因子：
4.8
通讯作者：
Huang, H. -C.
中科院分区：
文献类型：
--
作者：
Ferrari, Eric;He, Zhiqing;Huang, H. -C.
丙型肝炎病毒（HCV）NS 5 B蛋白是病毒RNA基因组复制所必需的RNA依赖性RNA聚合酶（RdRp）。据报道，纯化的NS 5 B在体外表现出多种活性。使用合成的杂聚RNA模板与双脱氧胞苷在其3 '端，我们检查从头开始和引物延伸的系统中没有自引发和末端核苷酸转移酶的活动。检测到主要是模板大小及其倍数的产物。发现对应于前三个掺入的核苷酸的高浓度的核苷三磷酸（K-m（app）类似于100 - 400 μ M）是有效的从头RNA合成所需的。然而，在起始二核苷酸或三核苷酸的存在下，达到最大活性所需的NTP的量下降了10（3）至10（4）倍，揭示了延伸所需的核苷酸大大减少（K-m（app）类似于0.03-0.09 μ M）。因此，在酶与模板和引物预孵育后从外源引物的单轮延伸也可以由以下支持：
The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K-m(app) similar to 100 - 400 mu M) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K-m(app) similar to 0.03-0.09 mu M). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by