NS5B与HCV负链RNA3′末端特异性结合的分析

Hepatitis C Virus (HCV) Encoded Non-structure Protein 5B Specifically Binds to the 3′-Terminal Sequences of Viral Negative-Strand RNA

NS5B是RNA依赖性RNA聚合酶 ,在病毒RNA合成过程中起到中心催化酶的作用 .在大肠杆菌中表达和提纯了GST NS5B融合蛋白 ,应用紫外交联试验 (UVcross linking)检测NS5B与丙型肝炎病毒 (HCV)负链RNA 3′末端的结合 ,确定NS5B是否参与HCV负链RNA 3′末端复制体的形成 .NS5B可与HCV负链RNA 3′末端发生结合 ,这种结合存在量效关系 ,比与正链RNA 3′UTRX区的结合强约 10倍 ,超大量的非同源性RNA和蛋白质不能竞争抑制NS5B与负链RNA 3′末端的结合 ,证明这种结合存在特异性 .结果提示NS5B是HCV负链RNA 3′末端复制体的成分之一 .

Hepatitis C virus(HCV) encoded non structure protein 5B(NS5B) is believed to be a RNA dependent RNA polymerase. GST NS5B fusion protein was expressed and purified and its ability to bind to the 1～585 nucleotides of 3′ terminal negative strand RNA sequences was examined by UV cross linking. Results presented here demonstrated that the NS5B binding to this region increased with the dosage of protein. The binding ability of NS5B to 3′ terminal negative strand RNA sequences was approximately 10 folds higher than to 3′ UTR X region of positive strand RNA. The specificity of NS5B binding to 3′ terminal negative strand RNA sequences was tested by competition with unlabelled RNA probe or an unrelated RNA/proteins. Results showed that the excess amount of cold probe RNA was able to almost completely compete out the complex resulted from protein RNA interaction. However unrelated RNA and protein were demonstrated no competition with NS5B. These results suggest that NS5B is a participating component of 3′ terminal replica of HCV negative strand RNA.