The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif
The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif
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DOI：
10.1074/jbc.ra118.005209
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发表时间：
2019-05-10
影响因子：
4.8
通讯作者：
Bressanelli, Stephane
中科院分区：
文献类型：
--
作者：
Ben Ouirane, Kaouther;Boulard, Yves;Bressanelli, Stephane
RNA病毒在受感染的宿主中合成新的基因组，这要归功于专用的病毒编码的RNA依赖性RNA聚合酶（RdRps）。因此，这些酶是抗病毒治疗的主要靶点，如最近针对丙型肝炎病毒（HCV）所证明的。然而，RdRps的结构和动力学的特殊性提出了关于在RNA聚合过程中进入其活性位点的基本问题。在这里，我们使用分子建模和分子动力学模拟，从可用的晶体结构的HCV NS5B与模板引物双链体和核苷酸的三元复合物，以解决核糖核苷酸进入病毒RdRp的活性位点的问题。跟踪可能通过传入的UTP或GTP通过RdRP特定的入口隧道，我们发现了两个连续的检查点，调节核苷酸流量的活性位点。我们观察到，镁结合的核苷酸首先结合旁边的隧道入口，并与三磷酸部分的相互作用定向，使其碱基部分进入第一。然后，RdRp基序F1 + F3的动力学允许核苷酸在核苷酸插入活性位点之前询问RNA模板碱基。这些动力学由第二镁二价阳离子精细调节，从而协调镁结合的核苷酸的进入与双金属离子催化所需的第二镁的穿梭。我们的研究结果表明，至少有一些这些功能是通用的病毒RdRps，并提供了更多的细节在RNA病毒的RdRps的原始核苷酸选择机制。
RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.