The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for Replication of MERS-CoV and SARS-CoV-2

  1. Natacha S. Ogando
  2. Jessika C. Zevenhoven-Dobbe
  3. Yvonne van der Meer
  4. Peter J. Bredenbeek
  5. Clara C. Posthuma
  6. Eric J. Snijder

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Abstract

The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

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  1. Version published to 10.1128/jvi.01246-20
  2. ScreenIT

    SciScore for 10.1101/2020.06.19.162529: (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.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Immunolabelling was performed as described before (69), using antibodies recognizing double-stranded RNA (dsRNA; (87)), M protein (52) or a cross-reacting antibody raised against SARS-CoV-nsp3 (88).
    SARS-CoV-nsp3 ( 88
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Infection of Vero, Vero E6 and HuH7 cells was carried out in EMEM containing 2 % FCS.
    Vero E6
    suggested: RRID:CVCL_XD71)
    HuH7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    5 μg of RNA was electroporated into 5×106 BHK-21 cells using the Amaxa nucleofector 2b (program A-031) and Nucleofection T solution kit (Lonza)
    BHK-21
    suggested: None
    In order to confirm the presence of engineered mutations in viral progeny, HuH7 and Vero cells were infected with supernatants harvested from transfected cells and intracellular RNA was isolated at 18 h post infection as described above.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    After hybridization, RNA bands were visualised (using exposure times of up to 28 days) and quantified by phosphorimaging using a Typhoon-9410 variable mode scanner (GE Healthcare) and ImageQuant TL software (GE Healthcare).
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    , 10 μM adenosyl-methionine (AdoMet, Thermofisher), 0.03 μCi/μl [3H]AdoMet (PerkinElmer) (25).
    Thermofisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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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  3. Version published to 10.1101/2020.06.19.162529v1 on bioRxiv