Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

核苷酸类似物抑制剂，包括广谱瑞姆昔韦和法维拉韦，在体外已显示出希望尽管对病毒RNA依赖的RNA聚合酶nsp12药物相互作用的机理尚不完全了解，但对于COVID-19治疗的检测方法和一些临床研究仍然如此。在这里，我们通过确定停滞的前和后易位聚合酶复合物的冷冻-EM结构来检查SARS-CoV-2 RNA复制的分子基础。与载脂蛋白复合物相比，该结构显示出nsp12及其辅因子nsp7和nsp8发生了显着的结构重排，以容纳核酸，而nsp12中存在高度保守的残基，从而将模板和引物定位在传入的核苷酸。此外，我们通过结构和动力学分析研究了雷姆昔韦的三磷酸代谢产物的抑制机制。