Identification of DAXX as a restriction factor of SARS-CoV-2 through a CRISPR/Cas9 screen

  1. Alice Mac Kain
  2. Ghizlane Maarifi
  3. Sophie-Marie Aicher
  4. Nathalie Arhel
  5. Artem Baidaliuk
  6. Sandie Munier
  7. Flora Donati
  8. Thomas Vallet
  9. Quang Dinh Tran
  10. Alexandra Hardy
  11. Maxime Chazal
  12. Françoise Porrot
  13. Molly OhAinle
  14. Jared Carlson-Stevermer
  15. Jennifer Oki
  16. Kevin Holden
  17. Gert Zimmer
  18. Etienne Simon-Lorière
  19. Timothée Bruel
  20. Olivier Schwartz
  21. Sylvie van der Werf
  22. Nolwenn Jouvenet
  23. Sébastien Nisole
  24. Marco Vignuzzi
  25. Ferdinand Roesch

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Abstract

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

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  1. Version published to 10.1038/s41467-022-30134-9
  2. Version published to 10.1101/2021.05.06.442916v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.05.06.442916: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: For Western Blot, we used mouse anti-DAXX (diluted 1:1000, Abnova #7A11),
    anti-DAXX
    suggested: None
    Secondary antibodies were goat anti-mouse and anti-rabbit HRP-conjugates (diluted 1:5000, GE Healthcare #NA931V and #NA934V).
    anti-mouse
    suggested: (GE Healthcare Cat# NA931, RRID:AB_772210)
    Secondary antibodies were goat anti-rabbit AF555 and anti-mouse AF488 (diluted 1:1000, ThermoFisher #A-21428 and #A-28175).
    anti-rabbit
    suggested: (Molecular Probes Cat# A-21428, RRID:AB_141784)
    For flow sorting of infected cells, we used the anti-S2 H2 162 antibody (diluted 1:150), a kind gift from Dr. Hugo Mouquet, (Institut Pasteur, Paris, France).
    anti-S2 H2 162
    suggested: None
    Secondary antibody was donkey anti-mouse AF647 (diluted 1:1000, Invitrogen #A31571).
    anti-mouse AF647
    suggested: None
    Secondary antibodies were goat anti-rat AF647 and anti-mouse AF488 (diluted 1:1000, ThermoFisher #A-21247 #A-28175).
    anti-rat AF647
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, viruses & plasmids: HEK 293T (ATCC #CRL-11268) were cultured in MEM (Gibco #11095080) complemented with 10% FBS (Gibco #A3160801) and 2 mM L-Glutamine (Gibco # 25030081).
    HEK 293T
    suggested: ATCC Cat# CRL-11268G-1, RRID:CVCL_UE07)
    VeroE6 (ATCC #CRL-1586), A549 (ATCC #CCL-185) and HEK 293T, both overexpressing the ACE2 receptor (A549-ACE2 and HEK 293T-ACE2, respectively), were grown in DMEM (Gibco #31966021) supplemented with 10% FBS (Gibco #A3160801), and penicillin/streptomycin (100 U/mL and 100 µg/mL, Gibco # 15140122).
    A549
    suggested: None
    HEK 293T-ACE2
    suggested: None
    Viral stocks were generated by infecting VeroE6 cells (MOI 0.01, harvesting at 3 dpi) using DMEM supplemented with 2% FBS and 1 μg/mL TPCK-trypsin (Sigma-Aldrich #1426-100MG).
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    CRISPR/Cas9 screen: 4×107 A549-ACE2 cells were treated with IFNα (200U/mL).
    A549-ACE2
    suggested: None
    Overexpression assay: 2×105 293T-ACE2 cells were seeded in a 24-well plate 18h before experiment.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Recombinant DNA
    SentencesResources
    The plentiCRISPRv.2 backbone was ordered through Addgene (Plasmid #52961). pMD2.G and psPAX2 were gifts from Didier Trono (Addgene #12259; #12260).
    plentiCRISPRv.2
    suggested: None
    pMD2 . G
    suggested: None
    psPAX2
    suggested: RRID:Addgene_12260)
    Cells were transfected with 500 ng of plasmids expressing HA-DAXX WT, HA-DAXX 15KR and HA-DAXXΔSIM plasmids, using Fugene 6 (Promega # E2691), following the manufacturer’s instructions.
    HA-DAXXΔSIM
    suggested: None
    Software and Algorithms
    SentencesResources
    The reference library was built using bowtie2 v2.2.9 (59).
    bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    Read mapping was performed with bowtie2 allowing 1 seed mismatch in --local mode and samtools v1.9 (60)
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Mapping analysis and gene selection were performed using MAGeCK v0.5.6, normalizing the data with default parameters.
    MAGeCK
    suggested: None
    10 pmol of NLS-Sp.Cas9-NLS (SpCas9) nuclease (Aldevron #9212) was combined with 30 pmol total synthetic sgRNA (10 pmol for each sgRNA) (Synthego) to form RNPs in 20 µL total volume with SE Buffer (Lonza #V5SC-1002).
    Aldevron
    suggested: (Aldevron, RRID:SCR_011017)
    Samples were acquired on a BD LSR Fortessa and analyzed using FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Image analysis was performed using ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analysis: GraphPad Prism was used for statistical analyses.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Linear models were computed using Rstudio.
    Rstudio
    suggested: (RStudio, RRID:SCR_000432)

    Results from OddPub: Thank you for sharing your code and 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 13. 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.

    About SciScore

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  4. Version published to 10.1101/2021.05.06.442916v1 on bioRxiv