Biochemical and structural analysis of the NS5B RNA‐dependent RNA polymerase of the hepatitis C virus
Biochemical and structural analysis of the NS5B RNA‐dependent RNA polymerase of the hepatitis C virus
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丙型肝炎病毒 NS5B RNA 依赖性 RNA 聚合酶的生化和结构分析
DOI：
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发表时间：
2000
影响因子：
2.5
通讯作者：
Bartenschlager
中科院分区：
文献类型：
--
作者：
Lohmann;Roos;Körner;Koch;Bartenschlager
丙型肝炎病毒（HCV）是黄病毒科病毒家族的一个独特成员，是全球慢性和散发性非甲非B型肝炎的主要病原体。这些病毒都有一个共同的正链RNA基因组，通过负链RNA中间体在感染细胞的细胞质中复制。由于缺乏可靠的细胞培养系统和方便的HCV动物模型，RNA复制的机制尚不清楚。作为开发合适的体外系统的第一步，我们在昆虫细胞中表达了NS5B RNA依赖性RNA聚合酶（RdRp），将蛋白质纯化至接近同质，并研究了其生化特性。它是一种引物和RNA模板依赖性RNA聚合酶，能够在没有额外的病毒或细胞辅因子的情况下复制长的杂聚模板。我们确定了最佳的反应参数，动力学常数和酶的底物特异性，这被证明是类似于脊髓灰质炎病毒的3D聚合酶所描述的。通过分析一系列核苷和非核苷化合物对RdRp活性的影响，我们发现三磷酸利巴韦林没有抑制作用，这提供了直接的实验证据，证明在患者中观察到的治疗效果与病毒聚合酶的直接抑制无关。最后，进行突变分析，以映射酶活性所需的最小NS5B序列，并鉴定对模板和NTP结合和催化重要的“经典”聚合酶基序。
Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non‐A, non‐B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus‐strand RNA genome that is replicated in the cytoplasm of the infected cell via minus‐strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA‐dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer‐ and RNA template‐dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular cofactors. We determined the optimal reaction parameters, the kinetic constants and the substrate specificity of the enzyme, which turned out to be similar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non‐nucleosidic compounds for their effect on RdRp activity, we found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase. Finally, mutation analysis was performed to map the minimal NS5B sequence required for enzymatic activity and to identify the ‘classical’ polymerase motifs important for template and NTP binding and catalysis.
影响因子：
3.7
作者：
Hwang, SB;Park, KJ;Lai, MMC
通讯作者：
Lai, MMC
影响因子：
3.7
作者：
Gale, MJ;Korth, MJ;Katze, MG
通讯作者：
Katze, MG
利巴韦林的分子作用机制。
DOI：
10.1093/clinids/12.6.1139
发表时间：
1990
期刊：
Reviews of infectious diseases
影响因子：
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作者：
Patterson,JL;Fernandez-Larsson,R
通讯作者：
Fernandez-Larsson,R