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

  1. Ruochen Zang
  2. James Brett Case
  3. Maria Florencia Gomez Castro
  4. Zhuoming Liu
  5. Qiru Zeng
  6. Haiyan Zhao
  7. Juhee Son
  8. Paul W. Rothlauf
  9. Gaopeng Hou
  10. Sayantan Bose
  11. Xin Wang
  12. Michael D. Vahey
  13. Tomas Kirchhausen
  14. Daved H. Fremont
  15. Michael S. Diamond
  16. Sean P.J. Whelan
  17. Siyuan Ding

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG) that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an ISG screen against VSV-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of virus replication. Mechanistically, internalized 25HC accumulates in the late endosomes and blocks cholesterol export, thereby restricting SARS-CoV-2 spike protein catalyzed membrane fusion. Our results highlight a unique antiviral mechanism of 25HC and provide the molecular basis for its possible therapeutic development.

Article activity feed

  1. 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

    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 10.1101/2020.06.08.141077 on bioRxiv