From the Laboratoire de Virologie Moléculaire et Structurale, CNRS UPR3296, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
CNRS UMR 52342, IFR66, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France.
J Biol Chem. 2010 Oct 22;285(43):32906-32918. doi: 10.1074/jbc.M110.151316. Epub 2010 Aug 20.
The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase essential for replication of the viral RNA genome. In vitro and presumably in vivo, NS5b initiates RNA synthesis by a de novo mechanism. Different structural elements of NS5b have been reported to participate in RNA synthesis, especially a so-called "β-flap" and a C-terminal segment (designated "linker") that connects the catalytic core of NS5b to a transmembrane anchor. High concentrations of GTP have also been shown to stimulate de novo RNA synthesis by HCV NS5b. Here we describe a combined structural and functional analysis of genotype 1 HCV-NS5b of strains H77 (subtype 1a), for which no structure has been previously reported, and J4 (subtype 1b). Our results highlight the linker as directly involved in lifting the first boundary to processive RNA synthesis, the formation of the first dinucleotide primer. The transition from this first dinucleotide primer state to processive RNA synthesis requires removal of the linker and of the β-flap with which it is shown to strongly interact in crystal structures of HCV NS5b. We find that GTP specifically stimulates this transition irrespective of its incorporation in neosynthesized RNA.
丙型肝炎病毒（HCV）NS5b 蛋白是一种 RNA 依赖的 RNA 聚合酶，对病毒 RNA 基因组的复制至关重要。在体外和推测的体内环境中，NS5b 通过从头（de novo）机制启动 RNA 合成。已有报道称 NS5b 的不同结构元件参与 RNA 合成，特别是所谓的“β-瓣”和连接 NS5b 催化核心与跨膜锚定的 C 端片段（称为“连接子”）。高浓度的 GTP 也被证明能刺激 HCV NS5b 的从头 RNA 合成。在这里，我们描述了对 H77（1a 型）和 J4（1b 型）两种基因型 1 HCV-NS5b 的结构和功能的综合分析。H77 型（1a 型）的 NS5b 结构此前尚未报道，而 J4 型（1b 型）的 NS5b 结构之前已有报道。我们的结果强调了连接子直接参与了提升至连续 RNA 合成的第一个边界的过程，形成了第一个二核苷酸引物。从第一个二核苷酸引物状态到连续 RNA 合成的转变需要去除连接子和β-瓣，晶体结构显示它们之间存在强烈相互作用。我们发现，GTP 特异性地刺激这种转变，而与它是否掺入新合成的 RNA 无关。
