Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit

We report an intersubunit interaction within the coronavirus replication-transcription complex that is critical for replication and evolutionarily conserved. We provide evidence that the nsp12-associated NiRAN domain has nucleoside monophosphate (NMP) transferase activity in trans and identified nsp9, an RNA-binding protein, as its target. NiRAN catalyzes the covalent attachment of an NMP moiety to the conserved nsp9 amino terminus in a reaction dependent on Mn²⁺ ions and an adjacent conserved Asn residue. NiRAN activity and nsp9 NMPylation were found to be essential for coronavirus replication. The data lead us to connect this activity of a nidovirus enzymatic marker with previous observations within a functionally and evolutionarily coherent hypothesis on the initiation of RNA synthesis in a class of RNA viruses.