Selection of 3′-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase
Selection of 3′-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase
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DOI：
10.1128/jvi.76.14.7030-7039.2002
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发表时间：
2002-07-01
影响因子：
5.4
通讯作者：
Hong, Z
中科院分区：
文献类型：
--
作者：
Shim, JH;Larson, G;Hong, Z
利用短RNA模板研究了丙型肝炎病毒（HCV）非结构蛋白5 B（NS 5 B）RNA依赖性RNA聚合酶的从头RNA合成。通过检查从头RNA合成的早期步骤来评估各种模板，包括来自HCV基因组的模板。NS 5 B显示能够从短至4-mer的模板和从HCV RNA基因组的正链和负链的3 '末端序列产生起始二核苷酸产物。GMP、GDP和鸟苷能够在从头RNA合成中充当起始核苷酸，表明起始核苷酸并不绝对需要三磷酸部分。在从头合成中积累了大量的起始产物，并且当大量的二核苷酸可用时，观察到从二核苷酸延伸。该结果表明，NS 5 B、模板和引入的核苷酸能够形成起始复合物，该起始复合物通过在转变为延伸复合物之前释放二核苷酸产物而频繁地中止。过渡是速率限制。此外，我们发现模板的二级结构对于从头起始并不重要，并且模板的Y末端碱基在选择起始位点时具有特异性。起始可以发生在从模板的3'端开始编号的+1、+2或+3位置，这取决于碱基组成。嘧啶碱基在三个位置中的任何一个都能够作为起始位点，而嘌呤碱基在+2和+3位置不支持起始。这一结果意味着HCV具有确保从头合成从+1位开始并保持其基因组3'端完整性的内在能力。该检测系统应该是一个重要的工具，调查从头启动的详细机制，HCV NS 5 B以及其他病毒RNA聚合酶。
De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3'-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that Y-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3' end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3' end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.