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

  1. Bing Wang
  2. Vladimir Svetlov
  3. Yuri I Wolf
  4. Eugene V Koonin
  5. Evgeny Nudler
  6. Irina Artsimovitch

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Abstract

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

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  1. Version published to 10.1101/2021.01.24.428004v2 on bioRxiv
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  3. Version published to 10.1101/2021.01.24.428004v1 on bioRxiv