The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus

  1. Natacha S. Ogando
  2. Jessika C. Zevenhoven-Dobbe
  3. Clara C. Posthuma
  4. Eric J. Snijder

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Abstract

Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless – in some cases – reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV.

I mportance

The bifunctional nsp14 subunit of the coronavirus replicase contains 3’-to-5’ exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 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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    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.

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  2. Version published to 10.1101/2020.06.19.162529 on bioRxiv