Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

  1. Florian Kabinger
  2. Carina Stiller
  3. Jana Schmitzová
  4. Christian Dienemann
  5. Goran Kokic
  6. Hauke S. Hillen
  7. Claudia Höbartner
  8. Patrick Cramer

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Abstract

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β- d - N 4 -hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

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  1. Version published to 10.1038/s41594-021-00651-0
  2. ScreenIT

    SciScore for 10.1101/2021.05.11.443555: (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

    Recombinant DNA
    SentencesResources
    Nsp12 protein was expressed from pFastBac vector 438C (Addgene #154759) in Hi5 insect cells.
    pFastBac
    suggested: RRID:Addgene_1925)
    Briefly, both proteins were expressed in E. coli BL21(DE3) RIL from the pET-derived vector 14-B (a gift from S. Gradia; Addgene 48308) in LB medium individually.
    pET-derived
    suggested: None
    Software and Algorithms
    SentencesResources
    The refined particles were then exported to RELION 3.156 and focus-refined in 3D with an initial local angular sampling of 3.7° and a mask around RdRp that omitted the nsp8 sliding poles and the upstream, second RNA turn.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Model quality was assessed using MolProbity within Phenix60 which revealed excellent stereochemistry for both structural models (Table 1).
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    Figures were prepared with PyMol (Schrödinger) and ChimeraX61.
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 16. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2021.05.11.443555v1 on bioRxiv