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![]() ![]() ![]() However, evidence to support this claim is lacking. Thus, it was presupposed that the CoV capping mechanism occurs in a similar manner to the eukaryotic capping pathway, with the NiRAN domain functioning as the GTase 3, 5, 26. In CoVs, the nsp13, nsp14 and nsp16 proteins have RTPase 25, N7-MTase 6 and 2′- O-MTase 7 activities, respectively. This canonical capping pathway, which is conserved among most viruses as well as all eukaryotes, entails (1) an RNA triphosphatase (RTPase), which removes the γ-phosphate from the nascent 5′-triphosphorylated RNA (5′-pppRNA) to yield a 5′-diphosphorylated RNA (5′-ppRNA) (2) a guanylyltransferase (GTase), which transfers GMP from GTP to 5′-ppRNA to form the core cap structure GpppN-RNA (3) a (guanine- N7)-methyltransferase ( N7-MTase), which methylates the cap guanine at the N7 position and (4) a (nucleoside-2′- O)-methyltransferase (2′- O-MTase), which methylates the ribose-2′-OH position on the first nucleotide of the RNA (Extended Data Fig. The 5′ cap structure was originally identified on RNAs of reovirus 21 and vaccinia virus 22, and the molecular mechanisms underlying the formation of the 5′ cap were subsequently determined in these systems 23, 24. Thus, formation of the RNA cap is crucial for successful replication and transcription of the viral genome. This 5′ cap is important for RNA stability, initiation of mRNA translation and protection from exonucleases 20. Methylation of the ribose 2′-OH position of the first nucleotide completes the cap and protects the RNA from the host immune system 18, 19. The CoV RNA genome, like eukaryotic mRNAs, contains a methylated guanosine linked to the first nucleotide of the RNA through a reverse 5′ to 5′ triphosphate linkage 1, 4. Several hypotheses for the function of the NiRAN domain have been proposed, including roles in protein-primed RNA synthesis, RNA ligation and mRNA capping 5, 17. 1) and are required for the replication of equine arteritis virus (EAV) and SARS-CoV-1 in cell culture 5. Notably, the active-site kinase-like residues of the NiRAN domain are highly conserved in Nidovirales (Extended Data Fig. The NiRAN domain shares sequence and structural similarity with the pseudokinase selenoprotein O (SelO), which transfers AMP from ATP to protein substrates (a process termed AMPylation) 14, 15, 16. nsp12 also contains an N-terminal NiRAN domain 5. At the core of the RTC is the nsp12 RNA-dependent RNA polymerase (RdRp), which is the target of antiviral agents used to treat COVID-19, including remdesivir 12 and molnupiravir 13. These polyproteins are cleaved by viral proteases to form 16 non-structural proteins (nsp1–16), some of which make up the replication–transcription complex (RTC) 2. The 5′ proximal two-thirds of the SARS-CoV-2 RNA genome contains two open reading frames that are translated by host ribosomes to form two large polyproteins 2. 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.Ĭoronaviruses (CoVs) are a family of positive-sense, single-stranded RNA viruses that cause disease in humans 9, including SARS-CoV-2, the aetiological agent of the ongoing COVID-19 pandemic 10, 11. Furthermore, we demonstrate in a reverse genetics system 8 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. Structural analyses of the replication–transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. The nsp14 6 and nsp16 7 methyltransferases then add methyl groups to form functional cap structures. Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain 5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA–protein intermediate (a process termed RNAylation). Here we reconstitute the N7- and 2′- O-methylated SARS-CoV-2 RNA cap ( 7MeGpppA 2′- O-Me) using virally encoded non-structural proteins (nsps). How this cap is made in SARS-CoV-2 is not completely understood. 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 response 1, 2, 3, 4. ![]()
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