Heparan sulfate assists SARS-CoV-2 in cell entry and can be targeted by approved drugs in vitro

  1. Qi Zhang
  2. Catherine Zhengzheng Chen
  3. Manju Swaroop
  4. Miao Xu
  5. Lihui Wang
  6. Juhyung Lee
  7. Amy Qiu Wang
  8. Manisha Pradhan
  9. Natalie Hagen
  10. Lu Chen
  11. Min Shen
  12. Zhiji Luo
  13. Xin Xu
  14. Yue Xu
  15. Wenwei Huang
  16. Wei Zheng
  17. Yihong Ye

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Abstract

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

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  1. Version published to 10.1038/s41421-020-00222-5
  2. ScreenIT

    SciScore for 10.1101/2020.07.14.202549: (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

    Experimental Models: Cell Lines
    SentencesResources
    Two ACE2-GFP cell lines using either HEK293 or HEK293T as the parental line were independently generated and used at NCATS and NIDDK, respectively.
    HEK293
    suggested: None
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    U2OS cells stably expressing Clathrin light chain-mCherry and Tractin-EGFP and the CRISPRv2 construct expressing sgRNA targeting SLC35B2 were described previously 19.
    U2OS
    suggested: None
    sgRNA-expressing lentiviruses were produced by transfecting 1 million 293FT cells (Thermo Fisher Scientific) in a 3.5 cm dish with 0.4 μg pVSV-G, 0.6 μg psPAX2, and 0.8 μg CRISPRv2-sgRNA
    293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    PP entry assay in the 1536-well format: HEK293-ACE2 cells seeded in white, solid bottom 1536-well microplates
    HEK293-ACE2
    suggested: None
    The plates were then spinoculated by centrifugation at 1500 rpm (453 x g) for 45 min and incubated for 24h (48 h for Calu-3 cells) at 37 °C 5% CO2 to allow cell entry of PP and the expression of luciferase.
    Calu-3
    suggested: None
    The plates were then incubated at 37 °C for 24 h (48 h for Calu3 cells) at 37 °C 5% CO2.
    Calu3
    suggested: None
    To detect the binding of SARS-CoV-2 to the cell surface, HEK293T-ACE2-GFP cells seeded in 24 well plates that had been coated with fibronectin were treated with 50 μl virus per well at 4 °C for 1 h with centrifugation at 1500 rpm (453 x g) for 60 min.
    HEK293T-ACE2-GFP
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were analyzed using MassLynx V4.1
    MassLynx
    suggested: (MassLynx , RRID:SCR_014271)
    Image processing and statistical analyses: Confocal images were processed using the Zeiss Zen software.
    Zeiss Zen
    suggested: None
    To measure fluorescence intensity, we used the Fiji software.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Data are presented as mean ± SEM, which was calculated by GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    p-values were calculated by Student’s t-test using Excel.
    Excel
    suggested: None
    Images were prepared with Adobe Photoshop and assembled in Adobe Illustrator.
    Adobe Photoshop
    suggested: (Adobe Photoshop, RRID:SCR_014199)
    Adobe Illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)

    Results from OddPub: Thank you for sharing your code.

    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 did not find any issues relating to the usage of bar graphs.

    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.

  3. Version published to 10.1101/2020.07.14.202549 on bioRxiv