ABSTRACT
Understanding the core replication complex of 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 NSP14, a bifunctional enzyme with an N-terminal 3′-to-5′ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and its accessory protein, NSP10. The difficulty in producing pure, 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 E. coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe 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-adenosyl homocysteine (SAH). Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analogue intermediate of remdesivir does not inhibit NSP14 activity; and second, SAH 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 COVID-19.
Fuente: Journal of Biological Chemistry
Available online 20 December 2021, 101518
