The RNA genome of SARS-CoV-2 contains a 5′ cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1–4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2′-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2′-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA–protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication–transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.
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