Substrate specificity of SARS-CoV-2 nsp10-nsp16 methyltransferase

  1. Roberto Benoni
  2. Petra Krafcikova
  3. Marek R. Baranowski
  4. Joanna Kowalska
  5. Evzen Boura
  6. Hana Cahová

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Abstract

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2) encodes all viral enzymes. In this work, we focus on one particular methyltransferase (MTase), nsp16, which in complex with nsp10 is capable of methylating the first nucleotide of a capped RNA strand at the 2′-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered five types of capped RNA (m 7 Gp 3 A(G)-, Gp 3 A(G)- and Gp 4 A-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for measurement of 2′-O-MTase activity. Further, we determined the IC 50 value of sinefungin (286 ± 66 nM) to illustrate the value of our approach for inhibitor screening. In the future, this approach can be used for screening inhibitors of any type of 2′-O-MTase.

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    SciScore for 10.1101/2020.07.30.228478: (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
    The digested RNA was purified using Amicon-Millipore filters 10 kDa (Merck) to get rid of Nuclease P1.
    Amicon-Millipore
    suggested: None
    Calculation of methylation efficiency: MassLynx software was used for data analysis and quantification of the relative abundance of all caps.
    MassLynx
    suggested: (MassLynx , RRID:SCR_014271)

    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

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  2. Version published to 10.1101/2020.07.30.228478v1 on bioRxiv