SUMMARY
The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), Nsp12, has a unique NiRAN domain that transfers nucleoside monophosphates to the Nsp9 protein. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation) Nsp12 exists in inactive state in which NiRAN/RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or NTPs partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN/RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically-linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and design of its inhibitors.
Highlights
Codon-optimization of Nsp12 triggers misfolding and activity loss
Slow translation, accessory Nsp7 and Nsp8 subunits, and NTPs rescue Nsp12
Non-substrate nucleotides activate RNA chain synthesis, likely via NiRAN domain
Crosstalk between two Nsp12 active sites that bind the same ligands
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Lead contact: Irina Artsimovitch, artsimovitch.1{at}osu.edu
In vitro data added indicating allosteric activation of SARS-CoV-2 RdRp by phosphorylated nucleotides, including remdesivir-TP, and ppGpp. Added discussion of evolutionary conservation of coronaviral genomes, allosteric regulation of RdRp activity by small molecules, and amino acid substitutions at/near domain interfaces.
