Mechanism of substrate binding by the SARS-CoV-2 NiRAN domain and modulation of its activities during replication

The SARS-CoV-2 Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain initiates viral genome capping by RNAylating nsp9 with the 5'-pppA-end of the genome followed by GDP-dependent deRNAylation to form the core capped GpppA-genome. Additionally, it cycles nsp9 through NMPylation-deNMPylation to generate GpppN. It is unclear how the distinct substrates, 5'-pppA-RNA and NTP, are bound, and how NiRAN balances RNAylation versus NMPylation. Earlier models proposed a common base-up pose for both the substrates. Here, structure-guided mutagenesis and reconstitution assays show that 5'-pppA indeed binds base-up during RNAylation, revealing that nsp12-Asp711 confers adenine selectivity, whereas, NTP adopts a perpendicular base-out pose during NMPylation. NiRAN intrinsically favors NMPylation over RNAylation, but nsp13 NTPase activity flips this preference. RNAylation weakens when RdRp is RNA-bound or replicating it, suggesting that a trans-acting NiRAN associated with an RNA-free RdRp performs capping. These findings provide insights into the orchestration of NiRAN activities and potential druggable sites for anti-viral therapeutics.