Motif V is an allosteric couple between the SARS-CoV-2 nsp13 nucleotide triphosphatase and helicase active sites

Non-structural protein 13 (nsp13), the Coronaviral RNA helicase, is an attractive antiviral target due to its importance in viral genome replication and highly conserved nature. Nsp13's processive dsRNA unwinding is driven by ATP hydrolysis in the nucleotide triphosphatase active site, which provides mechanical energy through conformational changes in the helicase domain to unidirectionally break hydrogen bonds between base pairs of bound dsRNA. Motif V is a conserved helical region between the nucleotide triphosphatase and helicase domains that has been previously shown to regulate ATP hydrolysis-mediated energy transduction within the flavivirus NS3 helicase. In this study, we characterized the role of the SARS-CoV-2 nsp13 Motif V in the regulation of ATPase-to-helicase crosstalk. Mutation of interacting Motif V residues T532 and S535 demonstrated increased rates of nucleic acid unwinding in an ATP-dependent manner, indicating the importance of the T532-S535 interaction in down-regulating energy transduction between the nucleotide triphosphatase and helicase domains. Furthermore, mutations to D534, which connects Motif V to the helicase domain through interaction with R560, severed the connection between the ATPase active site and the helicase domain via the disruption of a critical salt bridge. This connection was supported by the introduction of an L405D mutation, which attenuated helicase activity through repulsion of the D534 from R560. Overall, these data indicate that Motif V in SARS-CoV-2 nsp13 protein serves as a regulator of energy transduction in helicase function, similar to other families of positive-sense RNA viruses and helps define the mechanism of nsp13 helicase function.