SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors

  1. Chao Gao
  2. Junwei Zeng
  3. Nan Jia
  4. Kathrin Stavenhagen
  5. Yasuyuki Matsumoto
  6. Hua Zhang
  7. Jiang Li
  8. Adam J. Hume
  9. Elke Mühlberger
  10. Irma van Die
  11. Julian Kwan
  12. Kelan Tantisira
  13. Andrew Emili
  14. Richard D. Cummings

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Abstract

The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are highly expressed in innate immune cells in tissues susceptible to SARS-CoV-2 infection. Binding of the above receptors to S is characterized by affinities in the picomolar range and consistent with S glycosylation analysis demonstrating a variety of N- and O-glycans as receptor ligands. These results indicate multiple routes for SARS-CoV-2 to interact with human cells and suggest alternative strategies for therapeutic intervention.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.29.227462: (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
    Anti-Spike protein antibody (1A9), which was a mouse monoclonal antibody (IgG1) detecting the spike proteins of both SARS-CoV and SARS-CoV-2 through S2 subunit, was purchased from GeneTex (GTX632604).
    Anti-Spike protein
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    IgG1
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    The proteins were under the following concentrations: DC-SIGN, L-SIGN, Dectin-2, MGL all at 1 μg/ml, MR at 2.5 μg/ml, biotinylated plant lectins GNA and VVA both at 1 μg/ml, Con A at 0.1 μg/ml, or antibodies mAb100 at 1 μg/ml and anti-spike antibody 1A9 at 0.5 μg/ml.
    Dectin-2
    suggested: None
    anti-spike
    suggested: (Imported from the IEDB Cat# 1A9, RRID:AB_2848025)
    The cells were incubated with full-length S protein trimer (20 μg/ml) or the monoclonal antibody against DC-SIGN, #120507 (2 μg/ml) and L-SIGN, #120604 (2 μg/ml), or buffer only (negative control) on ice for 30 minutes.
    DC-SIGN
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells were transfected with MR-Fc DNA (kind gift from L. Martinez Pomares).
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Cell culture: Human fibroblast cell line 3T3, and the DC-SIGN-, and L-SIGN-transduced 3T3 cells (3T3-DC-SIGN+ and 3T3-DC-SIGNR+, respectively) were obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH from Drs.
    3T3
    suggested: None
    Vero E6 cells seeded in 6-well plates were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 1 or mock infected.
    Vero E6
    suggested: None
    Flow cytometry: For cell surface binding assay, the cultured 3T3, 3T3-DC-SIGN and 3T3-L-SIGN cells were collected and washed with cold PBS once.
    3T3-L-SIGN
    suggested: NIH-ARP Cat# 9948-54, RRID:CVCL_0R24)
    Software and Algorithms
    SentencesResources
    Cell culture: Human fibroblast cell line 3T3, and the DC-SIGN-, and L-SIGN-transduced 3T3 cells (3T3-DC-SIGN+ and 3T3-DC-SIGNR+, respectively) were obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH from Drs.
    AIDS Reagent Program
    suggested: None
    KewalRamani, HIV Drug Resistance Program, NCI45.
    Drug Resistance Program
    suggested: None
    Affinity constant was calculated with GraphPad Prism 6.0 (GraphPad Software, Inc.).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The results shown were from three independent experiments analyzed by FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    h5ad files were loaded by “read_h5ad” and violin plots were illustrated by “pl.stacked_violin” in scanpy 1.5.1, which is a model for single cell analysis in Python57.
    Python57
    suggested: None
    For the single-cell RNA sequencing analysis of bronchoalveolar immune cells in patients with SARS-CoV-2, dataset was retrieved from Liao et al.12 and Gene Expression Omnibus (GEO) under the accession number GSE145926, which contains 6 severe and 3 moderate SARS-CoV-2 patients and 3 healthy controls12.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    h5 format was loaded for analysis through R package Seurat v358, 59.
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)

    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

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  2. Version published to 10.1101/2020.07.29.227462v1 on bioRxiv