The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

  1. Wai Tuck Soh
  2. Yafei Liu
  3. Emi E. Nakayama
  4. Chikako Ono
  5. Shiho Torii
  6. Hironori Nakagami
  7. Yoshiharu Matsuura
  8. Tatsuo Shioda
  9. Hisashi Arase

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.11.05.369264: (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
    Antibodies: Mouse anti-CD209 mAb (clone 9E9A8), mouse IgG2a isotype control antibody (BioLegend, San Diego, CA, USA), mouse anti-L-SIGN mAb (clone 19F7), mouse anti-MHCII (clone WR18) (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-human ACE2 mAb (R&D Systems, Minneapolis, MN, USA), rat anti-Flag mAb (Sigma-Aldrich, St Louis, MI, USA), mouse anti-human CD14-APC mAb (eBioscience, San Diego, CA, USA), Donkey anti-mouse IgG-APC mAb, anti-human IgG-Fc fragment specific-APC mAb, anti-rat IgG-APC mAb (Jackson ImmunoResearch, West Grove, PA, USA).
    mouse IgG2a
    suggested: None
    anti-L-SIGN
    suggested: None
    anti-MHCII
    suggested: None
    anti-human ACE2
    suggested: None
    anti-Flag
    suggested: None
    anti-human CD14-APC
    suggested: None
    anti-mouse IgG-APC
    suggested: (SouthernBiotech Cat# 0107-11, RRID:AB_2793925)
    anti-human IgG-Fc
    suggested: None
    anti-rat IgG-APC
    suggested: (SouthernBiotech Cat# 0108-11, RRID:AB_2793937)
    The expression of CD209 was detected after two days of stimulation using an anti-CD209 monoclonal antibody.
    anti-CD209
    suggested: None
    For neutralization assay, essentially the same protocol was conducted with the exception that the pseudovirus was preincubated with diluted patient sera or anti-NTD, clone 4A8 and 4A2 monoclonal antibodies for 30 minutes at room temperature.
    anti-NTD
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and cell culture: The following cell lines were used in this study: Vero E6 is an African Green Monkey Kidney cell line, HEK293T cells are a human kidney cell line, HEK293T-DC-SIGN is a stable transfectant of HEK293T expressing DC-SIGN, HEK293T-L-SIGN is a stable transfectant of HEK293T expressing L-SIGN, HEK293T-ACE2 is a stable transfectant of HEK293T expressing ACE2, HEK293T-L-SIGN/TMPRSS2 is a stable transfectant of HEK293T expressing L-SIGN and TMPRSS2, HEK293T-DC-SIGN/TMPRSS2 is a stable transfectant of HEK293T expressing L-SIGN and TMPRSS2.
    HEK293T
    suggested: None
    The recombinant mAbs, C144, 4A2, and 4A8 were produced using Expi293 cells.
    Expi293
    suggested: RRID:CVCL_D615)
    After incubation, the cells were washed three times with DMEM and subsequently added to Vero cells at a 1:1 ratio.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    Cell lines and cell culture: The following cell lines were used in this study: Vero E6 is an African Green Monkey Kidney cell line, HEK293T cells are a human kidney cell line, HEK293T-DC-SIGN is a stable transfectant of HEK293T expressing DC-SIGN, HEK293T-L-SIGN is a stable transfectant of HEK293T expressing L-SIGN, HEK293T-ACE2 is a stable transfectant of HEK293T expressing ACE2, HEK293T-L-SIGN/TMPRSS2 is a stable transfectant of HEK293T expressing L-SIGN and TMPRSS2, HEK293T-DC-SIGN/TMPRSS2 is a stable transfectant of HEK293T expressing L-SIGN and TMPRSS2.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Flow cytometry analysis: FACSVerse™ or FACSCalibur flow cytometer (Becton Dickinson, Franklin Lake, NJ, USA) was used for flow cytometry analysis.
    FACSVerse™
    suggested: None
    FACSCalibur
    suggested: None
    3a was generated using the CLUSTAL Omega multiple sequence alignment tool from NCBI.
    CLUSTAL Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    FlowJo (BD Biosciences, San Jose, CA, USA) was used for analyzing flow cytometry data and Graphpad Prism version 7.0e was used for graph generation and statistical analysis.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Graphpad Prism
    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.
    • 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.

  2. Version published to 10.1101/2020.11.05.369264v1 on bioRxiv