Chen James, Malone Brandon, Llewellyn Eliza, Grasso Michael, Shelton Patrick M M, Olinares Paul Dominic B, Maruthi Kashyap, Eng Ed, Vatandaslar Hasan, Chait Brian T, Kapoor Tarun, Darst Seth A, Campbell Elizabeth A
Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY, 10065 USA.
Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, 10065 USA.
bioRxiv. 2020 Jul 13:2020.07.08.194084. doi: 10.1101/2020.07.08.194084.
SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是2019 - 2020年大流行的病原体。SARS-CoV-2基因组由RNA依赖性RNA聚合酶全酶（亚基nsp7/nsp82/nsp12）以及一系列辅助因子进行复制转录。其中一个因子是nsp13解旋酶。全酶RdRp和nsp13对病毒复制均至关重要，并且是治疗2019冠状病毒病（COVID-19）的靶点。在此，我们展示了SARS-CoV-2全酶RdRp与RNA模板 - 产物以及两个nsp13解旋酶分子复合物的冷冻电子显微镜结构。每个nsp13的尼多病毒目特异性N端结构域与每个nsp8拷贝的N端延伸相互作用。一个nsp13还与nsp12拇指结构域接触。该结构将解旋酶的核酸结合ATP酶结构域直接置于正在进行复制转录的全酶RdRp前方，限制了nsp13功能的模型。我们还观察到在nsp12 N端尼多病毒RdRp相关核苷酸转移酶结构域中结合有ADP - Mg2+，详细说明了一个用于抗病毒治疗开发的新口袋。
