Template/Primer Requirements and Single Nucleotide Incorporation by Hepatitis C Virus Nonstructural Protein 5B Polymerase

Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

丙型肝炎病毒（Hepatitis C Virus, HCV）的非结构蛋白5B（Nonstructural Protein 5B, NS5B）具有RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase）活性，负责介导病毒基因组RNA的复制。尽管已有多项针对这一关键病毒酶的表征研究，但由于缺乏可高效组装具有催化活性的聚合酶/模板/引物/核苷酸四元复合物的动力学系统，学界对NS5B催化的核苷酸掺入反应通路仍所知甚少。本研究针对HCV NS5B高效合成RNA所需的模板/引物条件展开了系统探究。对于分子内回折RNA合成而言，NS5B可利用3'端带有不稳定茎环结构、且在溶液中以单链分子形式存在的模板；而3'端携带稳定四核苷酸环的模板则无法支持HCV NS5B介导的RNA合成。基于上述实验观察，本研究合成了多款单链RNA模板，并搭配长度为2至5个核苷酸的短RNA引物开展了测试。结果显示，HCV NS5B可高效利用二核苷酸或三核苷酸起始RNA复制过程。进一步研究表明，聚合酶、模板与引物可在模板RNA的3'端组装形成具备起始活性的复合物，此时模板与引物可在聚合酶的活性位点空腔内形成碱基配对。模板的最小长度为5个核苷酸，这与NS5B/RNA复合物的结构模型相符——该模型显示，五核苷酸单链RNA模板占据了聚合酶手指结构域沿线的凹槽。这一发现提示，RNA在NS5B聚合酶上的初始锚定需要单链RNA分子。拇指结构域中一个独特的β发夹环（β-hairpin loop）可能在正确定位单链模板以起始RNA合成的过程中发挥重要作用。明确模板/引物的必要条件，将有助于推进HCV NS5B及其抑制剂的机制表征研究。