Allosteric activation of SARS-CoV-2 RdRp by remdesivir triphosphate and other phosphorylated nucleotides

Abstract

RNA-dependent RNA polymerase (RdRp) is a primary target for antivirals. We report that Nsp12, a catalytic subunit of SARS-CoV-2 RdRp, produces an inactive enzyme when codon-optimized for bacterial expression. We also show that accessory subunits, NTPs, and translation by slow ribosomes partially rescue Nsp12. Our findings have implications for functional studies and identification of novel inhibitors of RdRp and for rational design of other biotechnologically and medically important expression systems.

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

“…To match the previously published conditions, we carried GMPylation assays with Nsp12 alone. To ascertain that this activity is preserved in context of the transcribing RdRp holoenzyme (Nsp12•7•8 2 ), we repeated our assays in the presence of Nsp7, Nsp8, and an RNA scaffold under conditions that support robust RNA extension by SARS-CoV-RdRp (9,22). Our results demonstrate comparable Nsp9 modification by Nsp12 alone or as part of an active transcription complex ( Fig.…”

“…We speculate that differences in RdRp folding could explain Mn 2+ dependence. CoV RdRps are highly dynamic enzymes that undergo large conformational changes during the transcription cycle (35) and can become misfolded during expression in heterologous hosts (22). In particular, the NiRAN domain has been captured in different conformational states in cryoEM structures and becomes more ordered upon ligand binding to the active site (9,14,17,36,37).…”

“…Our findings that noncognate NTPs and nucleoside analogs modulate RdRp activity suggested that the RdRp and NiRAN active sites (thereafter referred to as AS1 and AS2, respectively) could be allosterically connected (22).…”

“…The expression and purification of Nsp7/8/12 and Nsp12 mutants are described in our previous study (22). All purification steps were carried out at 4 o C. Nsp9 variants were overexpressed in E. coli BL21 (DE3) cells (Novagen, Cat#69450).…”

“…Our findings that noncognate NTPs and nucleoside analogs modulate RdRp activity suggested that the RdRp and NiRAN active sites (thereafter referred to as AS1 and AS2, respectively) could be allosterically connected (22). Testing this hypothesis necessitates parallel assays of both nucleotidyl transfer activities and in turn requires using cognate NiRAN substrates.…”