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

Keith J Mickolajczyk, Patrick M M Shelton, Michael Grasso, Xiaocong Cao, Sara E Warrington, Amol Aher, Shixin Liu, Tarun M Kapoor

Biophysical journal 2021 Mar 16

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The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. 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 RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws 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. Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Citation

Keith J Mickolajczyk, Patrick M M Shelton, Michael Grasso, Xiaocong Cao, Sara E Warrington, Amol Aher, Shixin Liu, Tarun M Kapoor. Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase. Biophysical journal. 2021 Mar 16;120(6):1020-1030