ACE2-independent interaction of SARS-CoV-2 spike protein to human epithelial cells can be inhibited by unfractionated heparin

  1. Lynda J. Partridge
  2. Lucy Urwin
  3. Martin J.H. Nicklin
  4. David C. James
  5. Luke R. Green
  6. Peter N. Monk

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Abstract

The SARS-CoV-2 spike protein is known to bind to the receptor, ACE2, on the surface of target cells. The spike protein is processed by membrane proteases, including TMPRSS2, and either internalises or fuses directly with the cell, leading to infection. We have identified a human cell line that expresses both ACE2 and TMPRSS2, the RT4 urinary bladder transitional carcinoma, and used it to develop a proxy assay for viral interactions with host cells. A tagged recombinant form of the spike protein, containing both the S1 and S2 domains, interacted strongly with RT4 cells as determined by flow cytometry, whereas the S1 domain and the receptor binding domain (RBD) interacted weakly. S1S2 interaction was temperature dependent and increased sharply at 37°C, suggesting that processing of the intact spike protein is likely to be important in the interaction. S1S2 protein could associate with cells with a low dependence on ACE2 expression, while RBD required the presence of ACE2 for interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we used a flow cytometric assay to determine the effect of heparin on spike protein interaction with RT4 cells. Unfractionated heparin inhibited spike protein interaction with an IC 50 value of <0.05U/ml whereas two low molecular weight heparins were much less effective. A mutant form of the spike protein, lacking the Arg-rich region proposed to be a furin cleavage site, interacted very weakly with cells and had a lower affinity for unfractionated and lower molecular weight heparin than the wild type spike protein. This indicates that the furin cleavage site might also be a heparin binding site and potentially important in interactions with host cells. Taken together, our data suggest that heparin, particularly unfractionated forms, could be considered to reduce clinical manifestations of COVID-19 by inhibiting continuing viral infection.

Author Summary

Since the emergence of SARS-CoV-2 in 2019, the world has faced a vast public health crisis. SARS-CoV-2 associates with human cells through interaction of the viral spike protein with the host receptor, ACE2. In the absence of a vaccine, new treatments are required to reduce the morbidity and mortality of SARS-CoV-2. Here, we use a novel technique to demonstrate spike protein interactions with human cells with low levels of ACE2 at the cell surface, suggesting a secondary receptor. We demonstrate the importance of a new heparin-binding site within the viral spike protein for these interactions. We also found that unfractionated heparin was able to bind to the viral spike protein and therefore, potently inhibit viral spike protein interactions with human cells. Our data demonstrate that ACE2 is not absolutely required for spike protein interactions with human cells and furthermore, that unfractionated heparin should be considered as a treatment to reduce SARS-CoV-2 viral infection.

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  1. Version published to 10.1101/2020.05.21.107870v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.05.21.107870: (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
    Fondaparinux was purchased from Merck, UK. Goat anti-human ACE2 antibody AF933 (Biotechne), goat control IgG AB-108-C (Biotechne), rabbit anti-human TMPRSS2 (MBS9215011, Gentaur), rabbit IgG control (Biolegend) were used as per manufacturers’ instructions.
    anti-human ACE2
    suggested: None
    anti-human TMPRSS2
    suggested: None
    rabbit IgG
    suggested: None
    Cells were washed once and then incubated with the appropriate fluorescently labelled secondary antibody (anti-mouse polyvalent Ig-FITC (Merck) or anti-His6 HIS.H8 DyLight 488, (Invitrogen) for 30 min at 21°C.
    anti-mouse polyvalent Ig-FITC
    suggested: None
    anti-His6
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The A549 cell line was obtained from the European Collection of Animal Cell Cultures (ECACC) and cultured in DMEM supplemented with 10% FCS.
    A549
    suggested: None
    293TACE2 cells were kindly provided by Paul Bieniasz (The Rockefeller University, USA), cultured as described for 293T cells including selection with 5ug/ml blasticidin.
    293TACE2
    suggested: RRID:CVCL_YZ65)
    A human keratinocyte cell line, HaCaT (300493), was obtained from Cell Line Services (CLS GmbH, Germany) and routinely cultured in DMEM supplemented with 10% FCS.
    HaCaT
    suggested: CLS Cat# 300493/p800_HaCaT, RRID:CVCL_0038)
    The Caco2 cell line (ATCC® HTB-37), derived from a colorectal adenocarcinoma, was obtained from Dr. Michael Trikic (University of Sheffield, UK).
    Caco2
    suggested: ATCC Cat# HTB-37, RRID:CVCL_0025)
    Real time quantitative PCR (RT-qPCR): HaCaT, RT4, A549, 293T, 293TACE2 and Caco2 cell lines were cultured for 48hrs under standard media conditions and harvested using trypsin.
    293T
    suggested: RRID:CVCL_YZ65)
    Wild-type S1S2 (wt S1S2; Val16-Pro1213; Stratech UK) with a His6 tag at the C-terminus was expressed in baculovirus-insect cells, while S1-Fc (Val16-Arg685; Stratech UK) with a mouse IgG1 Fc region at the C-terminus was expressed in HEK293 cells.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  3. Version published to 10.1101/2020.05.21.107870v1 on bioRxiv