NMPylation and de-NMPylation of SARS-CoV-2 Nsp9 by the NiRAN domain

Nsp12, the catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), contains two active sites that catalyze nucleotidyl-monophosphate (NMP) transfer (NMPylation). RNA synthesis is mediated by the RdRp active site that is conserved among all RNA viruses and has been a focus of mechanistic studies and drug discovery. The second active site resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. Both catalytic reactions are essential for viral replication, but the mechanism and targets of NiRAN are poorly characterized. One recent study showed that NiRAN transfers NMP to the first residue of RNA-binding protein Nsp9. Another study reported a structure of SARS-CoV-2 replicase with an extended Nsp9 in the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 Nsp12 efficiently and reversibly NMPylates the native but not the extended Nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas a substitution of a catalytic RdRp Asp residue does not. NMPylation is inhibited by nucleotide analogs, pyrophosphate, and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We hypothesize that Nsp9 remodels both active sites of Nsp12 to support initiation of RNA synthesis by RdRp and subsequent capping of the product RNA by the NiRAN domain.