Characterization of SARS2 Nsp15 nuclease activity reveals it's mad about U

  1. Meredith N Frazier
  2. Lucas B Dillard
  3. Juno M Krahn
  4. Lalith Perera
  5. Jason G Williams
  6. Isha M Wilson
  7. Zachary D Stewart
  8. Monica C Pillon
  9. Leesa J Deterding
  10. Mario J Borgnia
  11. Robin E Stanley

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Abstract

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.

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  1. Version published to 10.1093/nar/gkab719
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    SciScore for 10.1101/2021.06.01.446181: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Beam-induced motion and drift were corrected using MotionCor2 (24) and aligned dose-weighted images were used to calculate CTF parameters using CTFFIND4 (25).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    CryoSPARC v2 (26) was used in all subsequent image processing.
    CryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    A combination of rigid body and real-space refinement in Phenix as well as iterative rounds of building in COOT (28) were used to improve the fit of the model.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Molprobity (29) was used to evaluate the model (Table 1).
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Prism (Graphpad) was used to calculate significant differences using Dunnett’s T3 multiple corrections test.
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)

    Results from OddPub: Thank you for sharing your data.

    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.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.06.01.446181v1 on bioRxiv