SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation

  1. David W Sanders
  2. Chanelle C Jumper
  3. Paul J Ackerman
  4. Dan Bracha
  5. Anita Donlic
  6. Hahn Kim
  7. Devin Kenney
  8. Ivan Castello-Serrano
  9. Saori Suzuki
  10. Tomokazu Tamura
  11. Alexander H Tavares
  12. Mohsan Saeed
  13. Alex S Holehouse
  14. Alexander Ploss
  15. Ilya Levental
  16. Florian Douam
  17. Robert F Padera
  18. Bruce D Levy
  19. Clifford P Brangwynne

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Abstract

Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

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  1. Version published to 10.7554/elife.65962 on eLife
  2. ScreenIT

    SciScore for 10.1101/2020.12.14.422737: (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 mycoplasma free at the beginning of the study.

    Table 2: Resources

    No key resources detected.

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Here, we show that in vitro SARS-CoV-2 spike and ACE2 cell co-culture assays overcome this limitation, and uncover a critical role for viral membrane composition in infection and formation of pathological syncytia. Our approach relies on a combination of high throughput screening, quantitative live cell imaging, and viral infection assays, all of which implicate cholesterol-rich regions of the plasma membrane in spike-mediated membrane fusion. Cholesterol is known to preferentially interact with certain membrane proteins, particularly those modified with specific lipid moieties (e.g. palmitic acid) (Levental et al., 2010; Martin, 2013; Sobocińska et al., 2017), together clustering into nanodomains which have been referred to as lipid rafts (Levental et al., 2020; Simons and Ikonen, 1997; Veatch and Keller, 2005). In the context of SARS-CoV-2 spike, such cholesterol-rich nanodomains could potentially facilitate the energetically-unfavorable process of lipid bilayer mixing (Heald-Sargent and Gallagher, 2012; Kim and Chen, 2019; Tenchov et al., 2006). However, given its lack of partitioning into ordered GPMV domains (Figure 6D), spike may form nanoscale clusters by a different mechanism. A favored model is that an accessible population of cholesterol, independent of sphingolipids and rafts (Das et al., 2014; Kinnebrew et al., 2019), interacts directly with spike trimers and mediates formation of higher-order protein-lipid assemblies (Figure 7H). Precedent for this model is provi...

    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 10.1101/2020.12.14.422737 on bioRxiv