The role of high cholesterol in SARS-CoV-2 infectivity

  1. Hao Wang
  2. Zixuan Yuan
  3. Mahmud Arif Pavel
  4. Sonia Mediouni Jablonski
  5. Joseph Jablonski
  6. Robert Hobson
  7. Susana Valente
  8. Chakravarthy B. Reddy
  9. Scott B. Hansen

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Abstract

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  1. Version published to 10.1016/j.jbc.2023.104763
  2. Version published to 10.1101/2020.05.09.086249v5 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.05.09.086249: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Housing, animal care and experimental procedures were consistent with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    hACE2 expression was confirmed by SARS2-PV entry assays and by immunofluorescence staining using mouse monoclonal antibody recognizing c-Myc.
    c-Myc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: Human embryonic kidney 293T (HEK293T) cells and HEK293T cells overexpressing human ACE2 (hACE2) were grown in Dulbecco‘s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin.
    HEK293T
    suggested: None
    293T-hACE2 cells were selected and maintained with medium containing puromycin (Sigma).
    293T-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Lungs were removed from C57BL/6J wild type and diet induced obesity (DIO) mice.
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Software and Algorithms
    SentencesResources
    Statistical analyses: All statistical calculations were performed in GraphPad Prism v6.0.
    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: 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.

  4. Version published to 10.1101/2020.05.09.086249v4 on bioRxiv
  5. ScreenIT

    SciScore for 10.1101/2020.05.09.086249: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementHousing , animal care and experimental procedures were consistent with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ACE2 receptor ( cyan ) labeled with ACE2 antibody has very little overlap with GM1 lipid rafts when blood serum is removed ( middle panel) .
    ACE2
    suggested: None
    To determine the amount of ACE2 positioned for viral entry and its dependence on cholesterol , we co-labeled ACE2 and GM1 lipid rafts in HEK293T cells with ACE2 antibody labelled with Cy3b and Alexa Fluor 647 conjugated fluorescent CTxB ( a GM1 specific toxin) , treated cells with apoE/serum , and imaged with super-resolution direct stochastical optical reconstruction microscopy ( dSTORM)32–35 . dSTORM is capable of visualizing nanoscale arrangements ( i.e. sub-100 nm diameter lipid domain structures36 ) in intact cellular membranes .
    GM1
    suggested: None
    hACE2 expression was confirmed by SARS2-PV entry assays and by immunofluorescence staining using mouse monoclonal antibody recognizing c-Myc .
    c-Myc
    suggested: None
    For labelling , cells were incubated with primary antibody ( anti-ACE2 antibody ( Abcam , #ab189168) , anti-Furin antibody ( Abcam , #ab3467 ) or anti-TMPRSS2 antibody [ EPR3861 ] ( Abcam , #ab92323) ) for 60 min in 5 % BSA / 0.05 % Triton / PBS at room temperature followed by 5 washes with 1 % BSA / 0.05 % Triton / PBS for 15 min each .
    anti-Furin
    suggested: (Abcam Cat# ab3467, AB_303828)
          <div style="margin-bottom:8px">
        <div><b>anti-TMPRSS2</b></div>
        <div>suggested: (Abcam Cat# ab92323, <a href="https://scicrunch.org/resources/Any/search?q=AB_10585592">AB_10585592</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibody ( donkey anti-rabbit cy3b and CTxB-647 ) was added in the same buffer as primary for 30 min at room temperature followed by 5 washes as stated above.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-rabbit</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T-hACE2 cells were selected and maintained with medium containing puromycin ( Sigma) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T-hACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were incubated with 10 µg/mL RBD peptide in cell culture medium overnight at 37°C in 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Andor iXon 897 EMCCD camera was used along with the Zen 10D software for image acquisition and processing .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Zen</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cholesterol signals were then graphed ( Mean ± s . e.m . ) and statistically analyzed ( one-way ANOVA ) with GraphPad Prism software (</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

    Results from Barzooka: We also 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 OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  6. Version published to 10.1101/2020.05.09.086249v3 on bioRxiv
  7. Version published to 10.1101/2020.05.09.086249v1 on bioRxiv
  8. Version published to 10.1101/2020.05.09.086249v2 on bioRxiv