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

  1. Keith J. Mickolajczyk
  2. Patrick M.M. Shelton
  3. Michael Grasso
  4. Xiaocong Cao
  5. Sara E. Warrington
  6. Amol Aher
  7. Shixin Liu
  8. Tarun M. Kapoor

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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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  1. ScreenIT

    SciScore for 10.1101/2020.07.31.231274: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Enzyme parameters KM, kcat and Hill coefficients (h) were determined by fitting the plot of initial rates (first ~150 s) against substrate concentration to the Michaelis-Menten equation or the Hill equation using Prism v.6.0 (GraphPad Software,
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Data were fit using a sigmoidal dose dependent equation:

    using Prism v.6.0 (GraphPad Software, Inc)

    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Single-molecule data were analyzed in MATLAB (Natick, MA).
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.07.31.231274 on bioRxiv