Lohmann V, Roos A, Körner F, Koch J O, Bartenschlager R
Institute for Virology, Johannes-Gutenberg University Mainz, Obere Zahlbacher Strasse 67, Mainz, 55131, Germany.
Virology. 1998 Sep 15;249(1):108-18. doi: 10.1006/viro.1998.9311.
The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.
对丙型肝炎病毒的RNA依赖性RNA聚合酶（RdRp）的生化特性进行了分析。用重组杆状病毒在昆虫细胞中表达了带有六组氨酸亲和标签的NS5B融合蛋白，并将其纯化至接近均一。纯化蛋白的酶活性受到KCl或高浓度NaCl的抑制，并且绝对依赖于Mg2+，Mg2+可被Mn2+替代。发现NS5B具有持续性，能够在22℃下以150-200个核苷酸/分钟的延伸速率复制长的杂聚模板。测定了所有四种核苷三磷酸和不同模板的动力学常数。对于对应于丙型肝炎病毒基因组最后319个核苷酸的杂聚RNA模板，UTP、GTP、ATP和CTP的Km值分别约为1.0、约0.5、约10和约0.3 microM。几种RdRp活性抑制剂和底物类似物的概况表明，该酶对核糖核苷5'-三磷酸有强烈偏好，并且与小RNA病毒的3Dpol非常相似。
