Al R H, Xie Y, Wang Y, Hagedorn C H
Department of Medicine, Genetics Program of the Winship Cancer Center, Emory University School of Medicine, Atlanta, GA 30322, USA.
Virus Res. 1998 Feb;53(2):141-9. doi: 10.1016/s0168-1702(97)00147-0.
The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients. Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication. In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity. This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template. The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30 degrees C. Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme. Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors.
丙型肝炎病毒（HCV）是一个重大的公共卫生问题，可在慢性感染患者中导致肝衰竭和肝细胞癌。我们的目标是在大肠杆菌中表达HCV 1a基因型的HCV非结构蛋白5B（NS5B），并开始研究其在HCV基因组复制中的作用。在本报告中，我们证明了一种氨基末端序列为ASMSYSWTG的重组NS5B蛋白具有RNA依赖性RNA聚合酶（RDRP）活性。这种重组酶在聚（U）聚合酶测定中具有活性，并且当使用珠蛋白mRNA作为模板时可产生模板大小的RNA产物。重组NS5B的聚合酶活性是引物依赖性的，并且在30℃孵育至少60分钟时具有活性。HCV NS5B羧基末端区域的缺失导致RDRP活性丧失，表明观察到的酶活性归因于全长重组酶。重组NS5B（RDRP）应有助于理解HCV复制机制和鉴定特异性酶抑制剂。
