SARS-CoV-2 comprehensive receptor profiling: mechanistic insight to drive new therapeutic strategies

  1. Sarah MV Brockbank
  2. Jo Soden
  3. Raquel Faba-Rodriguez
  4. Lyn Rosenbrier Ribeiro
  5. Catherine Geh
  6. Helen Thomas
  7. Jenni Delight
  8. Lucy Coverley
  9. W Mark Abbott
  10. Jim Freeth

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Abstract

Here we describe a hypothesis free approach to screen for interactions of SARS-CoV-2 spike (S) protein with human cell surface receptors. We used a library screening approach to detect binding interactions across one of the largest known panels of membrane-bound and soluble receptors, comprising 5845 targets, expressed recombinantly in human cells. We were able confirm and replicate SARS-CoV-2 binding to ACE2 and other putative coreceptors such as CD209 and CLEC4M. More significantly, we identified interactions with a number of novel SARS-CoV-2 S binding proteins. Three of these novel receptors, NID1, CNTN1 and APOA4 were specific to SARS-CoV-2, and not SARS-COV, with APOA4 binding the S-protein with equal affinity as ACE2. With this knowledge we may further understand the disease pathogenesis of COVID-19 patients and how infection by SARS-CoV-2 may lead to differences in pathology in specific organs or indeed the virulence observed in different ethnicities. Importantly we illustrate a methodology which can be used for rapid, unbiassed identification of cell surface receptors, to support drug screening and drug repurposing approaches for this and future pandemics.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.11.434937: (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
    Either before or after fixing the cells, 10 μg/mL SARS-CoV-2 S protein alone, or pre-incubated with AlexaFluor647-conjugated goat anti-human IgG Fcγ pAb (Jackson ImmunoResearch; detection antibody) at a 2:1 molar ratio (‘pre-incubation method’), was incubated with the cell microarray slides for 1 hour.
    anti-human IgG
    suggested: None
    Cell microarray slides were incubated before, after, or in the absence of cell fixation with 10 μg/mL SARS-CoV-2 S test protein, 10 μg/mL SARS-CoV S control protein, 1 μg/mL Rituximab biosimilar (human IgG1; Absolute Antibody, UK), or phosphate-buffered saline (PBS) only, each pre-incubated with AlexaFluor647-conjugated goat anti-human IgG Fcγ pAb at a 2:1 molar ratio, or treated sequentially with the same detection antibody.
    S control protein , 1
    suggested: None
    human IgG1
    suggested: None
    After 48 h, cells were treated with Accutase (Sigma-Aldrich, UK), pelleted by centrifugation, and resuspended into incubation buffer (PBS supplemented with 10% foetal calf serum) containing a dose range of 0 to 150 μg/mL SARS-CoV-2 S protein or SARS-CoV S protein, or 1 μg/mL Rituximab biosimilar (human IgG1; Absolute Antibody, UK).
    SARS-CoV S protein ,
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    , SARS-CoV-2 S and SARS-CoV S respectively, were produced in the HEK-293 cell system and were purified from the culture supernatants by capture with Ni Sepharose excel affinity resin (Cytiva) followed by size exclusion chromatography using a Superose 6 16/60 column in phosphate-buffered saline (PBS).
    HEK-293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Human HEK293 cells were grown over the vector microarray, leading to reverse transfection at each location.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Software and Algorithms
    SentencesResources
    Library hits (duplicate Alexa-Fluor647-positive spots) were captured by fluorescence imaging (GE Ettan DIGE imager), and analysed using ImageQuant software (GE Healthcare).
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    Dose titration curves were plotted, and EC50 values determined, using Prism GraphPad.
    GraphPad
    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 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.
    • No funding statement was detected.
    • 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 10.1101/2021.03.11.434937v1 on bioRxiv