Qin W, Yamashita T, Shirota Y, Lin Y, Wei W, Murakami S
Department Molecular Oncology, Cancer Research Institute and First Department of Internal Medicine, Kanazawa University, Kanazawa, Ishikawa, Japan.
Hepatology. 2001 Mar;33(3):728-37. doi: 10.1053/jhep.2001.22765.
The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication. To further understand the structure and functions of NS5B, we introduced a series of 27 clustered and 19 point substitution mutations within and outside the well-known motifs conserved among RdRP by alanine scanning and investigated effects of these mutants on enzymatic activity of NS5B. Surprisingly, most of the mutations (22 of 27 clustered mutants) do not affect RdRP activity at all, indicating that the side chains of the corresponding amino acid residues are dispensable for the catalytic activity. On the other hand, 4 mutants, cm20t, cm194t, cm2t, and cm3t, are defective in RdRP activity. By further analysis with point mutations within these regions, E18, Y191, C274, Y276, and H502 were determined to be critical for the RdRP activity. Y276 was also shown to be critical for RNA template/primer association, although 3 amino acid sequences were identified to be important for RNA template binding by RNA-filter binding assays. Finally, 4 discontinuous sequences of NS5B (aa139-145, aa149-155, aa 365-371, and aa 382-388) were found to be essential for binding to NS5A as determined by glutathione S-transferase (GST)-pull down assays using GST-NS5A and FLAG-NS5B expressed in cotransfected cells, and GST-pull down assay in vitro. In light of the crystal structure models of NS5B recently reported, our results indicate that the RdRP activity of NS5B requires the longer loop and the helix located at the distal of the thumb, which are unique among RdRPs as well as reverse transcriptases.
丙型肝炎病毒（HCV）非结构蛋白5B（NS5B）是一种依赖RNA的RNA聚合酶（RdRP），是HCV复制的核心催化酶。为了进一步了解NS5B的结构和功能，我们通过丙氨酸扫描在RdRP中保守的著名基序内外引入了一系列27个聚类突变和19个点替换突变，并研究了这些突变体对NS5B酶活性的影响。令人惊讶的是，大多数突变（27个聚类突变体中的22个）根本不影响RdRP活性，这表明相应氨基酸残基的侧链对于催化活性是可有可无的。另一方面，4个突变体cm20t、cm194t、cm2t和cm3t的RdRP活性有缺陷。通过对这些区域内的点突变进行进一步分析，确定E18、Y191、C274、Y276和H502对RdRP活性至关重要。Y276也被证明对RNA模板/引物结合至关重要，尽管通过RNA滤膜结合试验确定了3个氨基酸序列对RNA模板结合很重要。最后，通过在共转染细胞中表达的GST-NS5A和FLAG-NS5B进行谷胱甘肽S-转移酶（GST）下拉试验以及体外GST下拉试验，发现NS5B的4个不连续序列（aa139-145、aa149-155、aa 365-371和aa 382-388）对于与NS5A结合至关重要。根据最近报道的NS5B晶体结构模型，我们的结果表明，NS5B的RdRP活性需要位于拇指远端的较长环和螺旋，这在RdRP以及逆转录酶中是独特的。
