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bioRxiv · 10.1101/2020.07.31.231274

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Mickolajczyk, K. J.·Shelton, P. M. M.·Grasso, M.·Cao, X.·Warrington, S. R.·Aher, A.·Liu, S.·Kapoor, T. M.
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Abstract

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

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Mickolajczyk, K. J., Shelton, P. M. M., Grasso, M., Cao, X., Warrington, S. R., Aher, A., Liu, S., Kapoor, T. M.. 2020-07-31. Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase. https://doi.org/10.1101/2020.07.31.231274

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