Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading

Liming Yan; Yunxiang Yang; Mingyu Li; Ying Zhang; Litao Zheng; Ji Ge; Yucen C Huang; Zhenyu Liu; Tao Wang; Shan Gao; Ran Zhang; Yuanyun Y Huang; Luke W Guddat; Yan Gao; Zihe Rao; Zhiyong Lou

doi:10.1016/j.cell.2021.05.033

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading

Cell. 2021 Jun 24;184(13):3474-3485.e11. doi: 10.1016/j.cell.2021.05.033. Epub 2021 May 24.

Authors

Liming Yan¹, Yunxiang Yang², Mingyu Li³, Ying Zhang³, Litao Zheng⁴, Ji Ge⁵, Yucen C Huang³, Zhenyu Liu³, Tao Wang¹, Shan Gao³, Ran Zhang⁵, Yuanyun Y Huang⁶, Luke W Guddat⁷, Yan Gao⁸, Zihe Rao⁹, Zhiyong Lou¹⁰

Affiliations

¹ MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
² MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
³ MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China.
⁴ Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China; School of Life Sciences, Tsinghua University, Beijing, China.
⁵ School of Life Sciences, Tsinghua University, Beijing, China.
⁶ Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.
⁷ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.
⁸ Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China. Electronic address: yan-gao13@mails.tsinghua.edu.cn.
⁹ MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China; School of Life Sciences, Tsinghua University, Beijing, China; State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences and College of Pharmacy, Nankai University, Tianjin, China; National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Guangzhou Laboratory, Guangzhou, China. Electronic address: raozh@tsinghua.edu.cn.
¹⁰ MOE Key Laboratory of Protein Science, School of Medicine, Tsinghua University, Beijing, China; Guangzhou Laboratory, Guangzhou, China. Electronic address: louzy@mail.tsinghua.edu.cn.

Abstract

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (^7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Keywords: SARS-CoV-2; cryo-EM; mRNA capping; proofreading; replication-transcription complex.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
COVID-19 / virology
Coronavirus RNA-Dependent RNA Polymerase / genetics*
Exoribonucleases / genetics*
Humans
Methyltransferases / genetics*
RNA, Messenger / genetics
RNA, Viral / genetics
SARS-CoV-2 / genetics*
Sequence Alignment
Transcription, Genetic / genetics
Virus Replication / genetics

Substances

RNA, Messenger
RNA, Viral
Methyltransferases
Coronavirus RNA-Dependent RNA Polymerase
NSP12 protein, SARS-CoV-2
Exoribonucleases