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

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

The capping of mRNA and the proofreading plays 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 a 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.