Syncytia formation by SARS‐CoV‐2‐infected cells

  1. Julian Buchrieser
  2. Jérémy Dufloo
  3. Mathieu Hubert
  4. Blandine Monel
  5. Delphine Planas
  6. Maaran Michael Rajah
  7. Cyril Planchais
  8. Françoise Porrot
  9. Florence Guivel‐Benhassine
  10. Sylvie Van der Werf
  11. Nicoletta Casartelli
  12. Hugo Mouquet
  13. Timothée Bruel
  14. Olivier Schwartz

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Abstract

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  1. Version published to 10.15252/embj.2020106267
  2. ScreenIT

    SciScore for 10.1101/2020.07.14.202028: (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 AuthenticationContamination: All cell lines were routinely tested for mycoplasma and found negative.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: Mouse monoclonal antibodies (mAb): IFITM1 (#60074-1-Ig, Proteintech) 1:250 for FACS, and IF; IFITM2/3 (#66081-1-Ig, Proteintech) 1:250 for FACS, and IF; ACE2 (AC18F) (#AG-20A-0032-C100 – adipogen).
    IFITM1
    suggested: None
    IF
    suggested: None
    Rabbit polyclonal antibodies: FLAG-Tag DYKDDDDK (D6W5B) (#14793, Cell Signaling) 1:800 for FACS.
    FLAG-Tag DYKDDDDK
    suggested: (Acris Antibodies GmbH Cat# AP08111PU-N, RRID:AB_1616157)
    Goat polyclonal antibodies: ACE2 (AF933 – R&D).
    ACE2
    suggested: None
    SARS_Ssd3 702 antibody was used at 0.5 μg/ml for FACS and IF. Human Mab: Anti-SARS-CoV2 monoclonal antibodies 48 and 71 recognize the S1 and S2 domains of the S protein, respectively (Planchais et al, manuscript in preparation).
    Anti-SARS-CoV2
    suggested: None
    The following antibodies were diluted in WB-buffer (PBS, 1% BSA, 0.05% Tween, 0.01% Na Azide): goat anti-human ACE2 (R&D cat#AF933, 1:2000), mouse anti-human ACE2 (Adipogen AC18F cat #AG-20A0032-C100, 1:1000), rabbit anti-human TMPRSS2 (Atlas antibodies cat# HPA035787, 1:1000), mouse anti-Flag tag (Sigma cat# F1804, 1:1000), rabbit anti-human actin (Sigma cat#A2066, 1:2000), mouse ascite anti-SARS S, (1:1000) (Siu, 2008 #4573).
    anti-human ACE2
    suggested: (Enzo Life Sciences Cat# ALX-804-715B-C050, RRID:AB_2050611)
    anti-human TMPRSS2
    suggested: None
    anti-Flag tag
    suggested: None
    anti-human actin
    suggested: (AMSBIO Cat# MA1000, RRID:AB_10920236)
    anti-SARS S,
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lentiviral and Retroviral vectors: For lentiviral production, 293T cells were co-transfected with pLenti6 or pLV derived vectors, packaging plasmid R8-2 and VSV-G plasmid as previously described 42.
    293T
    suggested: None
    GFP-Split fusion assay: For fusion assays with S-expressing cells, 293T-GFP1-10 and 293T-GFP11 cells (6×104 cells/well cells mixed at a 1:1 ratio) in 96 well plates (μClear, #655090) were transfected in suspension using Lipofectamine2000 (Thermo) with 100 ng of DNA. 10 ng of phCMV-SARS-CoV2-S, 25 ng of pCDNA3.1-hACE2, 25 ng of pCSDest-TMPRSS2 and 40 ng of pQCXIP-Empty or pQCXIP-IFITM-N-FLAG were used and adjusted to 100 ng DNA with pQCXIP-Empty.
    293T-GFP11
    suggested: None
    For donor cells, 293T-GFP1-10 cells were transfected with 10 ng of phCMV-SARS-CoV2-S, ±10 ng pCSDest-TMPRSS2, ±20 ng pQCXIP-IFITM-N-FLAG and adjusted to 50 ng with pQCXIP-Empty.
    293T-GFP1-10
    suggested: None
    U2OS viral mediated cell-cell fusion, video microscopy and immunofluorescence: U2OS-ACE2 GFP1-10 or GFP11 stably expressing TMPRSS2, IFITM1, 2, 3 or control cells were mixed (1:1 ratio) and plated at 8×103 cells per well in a 96 well plate (μClear, #655090), 24 h before infection.
    U2OS
    suggested: None
    Titration of viral stocks was performed on Vero E6, with a limiting dilution technique allowing a calculation of DCP50.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: pQCXIP-Empty control plasmid and pQCXIP-IFITM1-N-FLAG, pQCXIP-IFITM2-N-FLAG, pQCXIP-IFITM3-N-FLAG plasmids were described 42. pQCXIP-BSR-GFP11 and pQCXIP-GFP1-10 were from Yutaka Hata 51 (Addgene plasmid #68716; http://n2t.net/addgene:68716; RRID:Addgene_68716 and Addgene plasmid #68715; http://n2t.net/addgene:68715; RRID:Addgene_68715).
    detected: RRID:Addgene_68716)
    detected: RRID:Addgene_68715)
    pcDNA3.1-hACE2 was from Hyeryun Choe 52 (Addgene plasmid # 1786; http://n2t.net/addgene:1786; RRID:Addgene_1786).
    detected: RRID:Addgene_1786)
    pCSDest-TMPRSS2 was from RogerReeves 53 (Addgene plasmid # 53887; http://n2t.net/addgene:53887; RRID:Addgene_53887).
    detected: RRID:Addgene_53887)
    pLenti6-H2B-mCherry was from Torsten Wittmann 54 (Addgene plasmid # 89766; http://n2t.net/addgene:89766; RRID:Addgene_89766).
    detected: RRID:Addgene_89766)
    Software and Algorithms
    SentencesResources
    The GFP area was quantified on ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Images were quantified and processed using ImageStudioLite software.
    ImageStudioLite
    suggested: None
    Statistical analysis: Flow cytometry data were analyzed with FlowJo v10 software (TriStar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Figures were drawn on Prism 8 (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical analysis was conducted using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 13, 14, 16, 22, 23 and 24. 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.

  3. Version published to 10.1101/2020.07.14.202028v1 on bioRxiv