Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex
Structural basis for backtracking by the SARS-CoV-2 replication-transcription complex
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DOI：
10.1101/2021.03.13.435256
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发表时间：
2021-05-11
影响因子：
11.1
通讯作者：
Campbell, Elizabeth A.
中科院分区：
文献类型：
--
作者：
Malone, Brandon;Chen, James;Campbell, Elizabeth A.
回溯，即转录酶在核酸模板上的反向运动，是细胞生物中转录的普遍调控特征，但其在病毒中的作用尚未确定。在这里，我们提供的证据表明回溯延伸到了病毒领域，其中严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) RNA 依赖性 RNA 聚合酶 (RdRp) 的回溯可能有助于病毒转录和复制。 SARS-CoV-2 RdRp 与必需的 nsp13 解旋酶和 RNA 结合的结构表明解旋酶有助于回溯。我们使用冷冻电子显微镜、RNA-蛋白质交联和无偏分子动力学模拟来表征 SARS-CoV-2 RdRp 回溯。结果表明，回溯产生的产物 RNA 的单链 3' 片段通过 RdRp 核苷三磷酸 (NTP) 进入通道挤出，产物 RNA 3' 端的不匹配核苷酸磨损并进入 NTP 进入通道以启动回溯，并且 nsp13 刺激 RdRp 回溯。回溯可能有助于校对，这是 SARS-CoV-2 抗病毒药物耐药性的关键过程。
Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3 ' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3 ' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.