NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

Abstract

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme

“…The function of nsp9 nucleotidylation is less clear, with current studies proposing its involvement in either protein-primed RNA synthesis or the regulation of m 7 G cap synthesis. Nsp9 nucleotidylation is also not dependent on the nsp12 RdRp domain, possibly because RNA binding is not required for this reaction ( 18 , 20 ). Interestingly, all of these proposed activities utilise the same nsp12 D218 amino acid residue as the GTase reaction, raising the possibility that the NiRAN domain uses a single active site to carry out several diverse functions in coronavirus RNA synthesis (Figure 3G ) ( 19 , 20 ).…”

Reductionism Ad Absurdum: The Misadventures of Structural Biology in the Time of Coronavirus

“…The fact that nsps are formed by autocatalytic cleavage of polyproteins would suggest that the identities of their terminal residues are perhaps physiologically important. Indeed, we and others showed that the very first residue of nsp9 is NMPylated by NiRAN; 8 , 22 consequently, the native N-terminus is absolutely essential for nsp9 modification and viral replication in turn. 8 We also showed that AS2 can transfer RDV-monophosphate to nsp9; 22 an nsp9 thus mis-modified may be unable to support the viral life cycle.…”

“…Indeed, we and others showed that the very first residue of nsp9 is NMPylated by NiRAN; 8 , 22 consequently, the native N-terminus is absolutely essential for nsp9 modification and viral replication in turn. 8 We also showed that AS2 can transfer RDV-monophosphate to nsp9; 22 an nsp9 thus mis-modified may be unable to support the viral life cycle. While the exact role of nsp9 is yet unknown, its RNA-binding activity is thought to be critical.…”

“…While the exact role of nsp9 is yet unknown, its RNA-binding activity is thought to be critical. 8 , 11 , 22 Another recent study 23 concluded that nsp9-RNA contacts are distinct from those proposed based on the cryo-EM data analysis, 11 emphasizing the need for functional validation of structure-based models.…”

“… 4 , 11 Indeed, we found that nsp9 NMPylation is inhibited by nucleoside mono-, di-, and tetraphosphates, which cannot be used as substrates and are thus less likely to interfere with host enzymes. 22 …”