1. Academic Validation
  2. Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors

Activation of the SARS-CoV-2 NSP14 3'-5' exoribonuclease by NSP10 and response to antiviral inhibitors

  • J Biol Chem. 2022 Jan;298(1):101518. doi: 10.1016/j.jbc.2021.101518.
Amanda A Riccio 1 Eric D Sullivan 1 William C Copeland 2
Affiliations

Affiliations

  • 1 Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA.
  • 2 Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA. Electronic address: copelan1@niehs.nih.gov.
Abstract

Understanding the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the development of novel coronavirus-specific Antiviral therapeutics. Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional Enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in kcat/Km in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.

Keywords

COVID-19; NSP10; NSP14; SARS-CoV-2; coronavirus; enzyme kinetics; exoribonuclease; protein complex; protein drug interaction; protein purification; protein stability; protein–protein interaction; viral proofreading; viral protein.

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