Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase.
Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase.
复制标题
DOI：
10.1261/rna.031914.111
复制
发表时间：
2012-08
期刊：
影响因子：
4.5
通讯作者：
Robert C Vaughan;B. Fan;J. You;C. Kao
中科院分区：
文献类型：
--
作者：
Robert C Vaughan;B. Fan;J. You;C. Kao
了解丙型肝炎病毒(丙型肝炎病毒)依赖RNA的RNA聚合酶(RdRp)如何与新生RNA相互作用将为了解病毒的RNA合成机制提供有价值的见解。使用多肽质量指纹图谱方法和与烷基标记的新生RNA可逆交叉连接的多肽的亲和捕获，我们确定了丙型肝炎病毒RdRp指状域中Δ1环下方的一个区域，该区域与新生RNA接触。使用修饰保护试验来确认分配。在假定的新生RNA结合区内产生了几个突变，并分析了在体外和在丙型肝炎病毒亚基因组复制子中的RNA合成。在该区域内测试的所有突变都表明，依赖于引物的RNA合成减少，三元复合体的稳定性降低。这一研究结果加深了我们对丙型肝炎病毒RdRp的结构和功能以及对丙型肝炎病毒RNA合成的要求的理解。此外，还将新生RNA相互作用的模型与结构研究的结果进行了比较。
Understanding how the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) interacts with nascent RNA would provide valuable insight into the virus's mechanism for RNA synthesis. Using a peptide mass fingerprinting method and affinity capture of peptides reversibly cross-linked to an alkyn-labeled nascent RNA, we identified a region below the Δ1 loop in the fingers domain of the HCV RdRp that contacts the nascent RNA. A modification protection assay was used to confirm the assignment. Several mutations within the putative nascent RNA binding region were generated and analyzed for RNA synthesis in vitro and in the HCV subgenomic replicon. All mutations tested within this region showed a decrease in primer-dependent RNA synthesis and decreased stabilization of the ternary complex. The results from this study advance our understanding of the structure and function of the HCV RdRp and the requirements for HCV RNA synthesis. In addition, a model of nascent RNA interaction is compared with results from structural studies.