Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Cell (2020-06-12)
Quan Wang, Jiqin Wu, Haofeng Wang, Yan Gao, Qiaojie Liu, An Mu, Wenxin Ji, Liming Yan, Yan Zhu, Chen Zhu, Xiang Fang, Xiaobao Yang, Yucen Huang, Hailong Gao, Fengjiang Liu, Ji Ge, Qianqian Sun, Xiuna Yang, Wenqing Xu, Zhijie Liu, Haitao Yang, Zhiyong Lou, Biao Jiang, Luke W Guddat, Peng Gong, Zihe Rao
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, Grade I, ≥99%, from microbial
Sigma-Aldrich
Guanosine 5′-triphosphate sodium salt hydrate, ≥95% (HPLC), powder
Sigma-Aldrich
Spermidine trihydrochloride, ≥98% (TLC)
Sigma-Aldrich
Cytidine 5′-triphosphate disodium salt, ≥95%