The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signalling

  1. Lillian Cristina Russo
  2. Rebeka Tomasin
  3. Isaac Araújo Matos
  4. Antonio Carlos Manucci
  5. Sven T. Sowa
  6. Katie Dale
  7. Keith W. Caldecott
  8. Lari Lehtiö
  9. Deborah Schechtman
  10. Flavia Carla Meotti
  11. Alexandre Bruni-Cardoso
  12. Nicolas Carlos Hoch

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Abstract

SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.06.438552: (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 AuthenticationAuthentication: For most experiments, 6×6 adjacent fields of view were acquired per sample using automated autofocus and image acquisition settings.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primers and Antibodies: All oligonucleotides and antibodies used in this study are presented in the tables below:
    Antibodies
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293 FT cells were grown in DMEM/high glucose supplemented with 10% FBS (Thermo), 50mg/mL gentamycin (Sigma), 1mM sodium pyruvate (Thermo), non essential amino acids (Thermo) and 2mM L-glutamine (Thermo).
    HEK293
    suggested: None
    These vectors were co-transfected with psPAX2 and pMD2.G packaging vectors (Systems Biosciences) into HEK-293FT cells using standard PEI transfection, the supernatant collected 48h and 72h after transfection, filtered through 0.45 μm filters and used to transduce A549 cells in the presence of 8 μg/mL polybrene (Sigma).
    HEK-293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Software and Algorithms
    SentencesResources
    HEK293 FT cells were grown in DMEM/high glucose supplemented with 10% FBS (Thermo), 50mg/mL gentamycin (Sigma), 1mM sodium pyruvate (Thermo), non essential amino acids (Thermo) and 2mM L-glutamine (Thermo).
    Thermo
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    Molecular docking simulations were performed with Gold 5.4 and AutoDock 4.2.3 softwares.
    AutoDock
    suggested: (AutoDock, RRID:SCR_012746)
    Signals were quantified using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    This cDNA was used for qPCR (5 ng/reaction) using Power SYBR green Master mix (Thermo) with three technical replicates per biological replicate, using 200 nM of the primer sets indicated in Table 1, which were chosen from PrimerBank (Wang et al, 2012).
    PrimerBank
    suggested: (PrimerBank, RRID:SCR_006898)
    All graphs and statistical analyses were generated using GraphPad Prism software and display mean ± SEM of the normalized values relative to the IFNγ-treated control for each replicate, considered as 100%.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 26. 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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.04.06.438552 on bioRxiv