Substrate Specificity and Kinetics of RNA Hydrolysis by SARS-CoV-2 NSP10/14 Exonuclease

[Image: see text] Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19, continues to evolve resistance to vaccines and existing antiviral therapies at an alarming rate, increasing the need for new direct-acting antiviral drugs. Despite significant advances in our fundamental understanding of the kinetics and mechanism of viral RNA replication, there are still open questions regarding how the proofreading exonuclease (NSP10/NSP14 complex) contributes to replication fidelity and resistance to nucleoside analogs. Through single turnover kinetic analysis, we show that the preferred substrate for the exonuclease is double-stranded RNA without any mismatches. Double-stranded RNA containing a 3′-terminal remdesivir was hydrolyzed at a rate similar to a correctly base-paired cognate nucleotide. Surprisingly, single-stranded RNA or duplex RNA containing a 3′-terminal mismatch was hydrolyzed at rates 125- and 45-fold slower, respectively, compared to the correctly base-paired double-stranded RNA. These results define the substrate specificity and rate of removal of remdesivir for the exonuclease and outline rigorous kinetic assays that could help in finding next-generation exonuclease inhibitors or nucleoside analogs that are able to evade excision. These results also raise important questions about the role of the polymerase/exonuclease complex in proofreading during viral replication. Addressing these questions through rigorous kinetic analysis will facilitate the search for desperately needed antiviral drugs to combat COVID-19.