Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

  1. Ruochen Zang
  2. James Brett Case
  3. Eylan Yutuc
  4. Xiucui Ma
  5. Sheng Shen
  6. Maria Florencia Gomez Castro
  7. Zhuoming Liu
  8. Qiru Zeng
  9. Haiyan Zhao
  10. Juhee Son
  11. Paul W. Rothlauf
  12. Alex J. B. Kreutzberger
  13. Gaopeng Hou
  14. Hu Zhang
  15. Sayantan Bose
  16. Xin Wang
  17. Michael D. Vahey
  18. Kartik Mani
  19. William J. Griffiths
  20. Tom Kirchhausen
  21. Daved H. Fremont
  22. Haitao Guo
  23. Abhinav Diwan
  24. Yuqin Wang
  25. Michael S. Diamond
  26. Sean P. J. Whelan
  27. Siyuan Ding

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Abstract

The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease-2019 (COVID-19), has swept the world in unprecedented speed. In a few months, SARS-CoV-2 has infected millions of people and caused tens of thousands of deaths. There are no Food and Drug Administration-approved antivirals or vaccines yet available and clinical treatments are limited to supportive therapies that help alleviate the symptoms. Thus, there is an urgent need to identify effective antivirals as countermeasures before safe and effective vaccines are developed, tested, and then produced on a large scale. Our approach is to harness the germline-encoded interferon antiviral response to inhibit SARS-CoV-2 replication thereby limiting its pathogenicity.

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  1. Version published to 10.1073/pnas.2012197117
  2. ScreenIT

    SciScore for 10.1101/2020.06.08.141077: (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
    Cells were permeabilized and stained with antibodies against DAPI (P36962, Thermo Fisher), LAMP1 (9091S, Cell Signaling), LBPA (MABT837, Sigma), and Rab4 (ab13252, Abcam).
    antibodies against DAPI (P36962
    suggested: None
    LAMP1
    suggested: (Cell Signaling Technology Cat# 9091, RRID:AB_2687579)
    Rab4
    suggested: (Abcam Cat# ab13252, RRID:AB_2269374)
    Proteins were resolved in SDS-PAGE and analyzed by antibody as described (45) using the following antibodies and dilutions: GAPDH (631402, Biolegend), GFP (2555S, Cell Signaling), SARS-CoV-2 S1 (40590-T62, Sino Biological), SARS-CoV-2 S2 (40592-T62, Sino Biological), and V5 (13202S, Cell Signaling).
    GAPDH
    suggested: (BioLegend Cat# 631402, RRID:AB_2107422)
    GFP
    suggested: (Cell Signaling Technology Cat# 2555, RRID:AB_10692764)
    V5 (13202S, Cell Signaling).
    suggested: None
    Secondary antibodies were anti-rabbit (#7074, Cell Signaling) or anti-mouse (#7076, Cell Signaling) immunoglobulin G horseradish peroxidase-linked antibodies.
    anti-rabbit (#7074, Cell Signaling)
    suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)
    anti-mouse
    suggested: (Cell Signaling Technology Cat# 7076, RRID:AB_330924)
    Experimental Models: Cell Lines
    SentencesResources
    Flow cytometry: HEK293-hACE2 or HEK293-hACE2-TMPRSS2 cells with or without CH25H expression were inoculated with wild-type VSV-GFP, VSV-SARS-CoV, or VSV-SARS-CoV-2 at an MOI = 10 (based on titers in Vero cells) for 1 hr at 37°C.
    HEK293-hACE2-TMPRSS2
    suggested: None
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    GFP positive cells were determined by BD LSRFortessa™ X-20 cell analyzer and analyzed by FlowJo v10.6.2 (BD).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Images were visualized by Volocity v6.3 and quantification was determined by ImageJ (NIH).
    Volocity
    suggested: (Volocity 3D Image Analysis Software, RRID:SCR_002668)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    1E, 3B, S1C, S1D, and S2B was calculated by Student’s t test using Prism 8.4.2 (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    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 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:
    • 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

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  3. Version published to 10.1101/2020.06.08.141077v1 on bioRxiv