Neuropilin-1 is a host factor for SARS-CoV-2 infection

  1. James L. Daly
  2. Boris Simonetti
  3. Katja Klein
  4. Kai-En Chen
  5. Maia Kavanagh Williamson
  6. Carlos Antón-Plágaro
  7. Deborah K. Shoemark
  8. Lorena Simón-Gracia
  9. Michael Bauer
  10. Reka Hollandi
  11. Urs F. Greber
  12. Peter Horvath
  13. Richard B. Sessions
  14. Ari Helenius
  15. Julian A. Hiscox
  16. Tambet Teesalu
  17. David A. Matthews
  18. Andrew D. Davidson
  19. Brett M. Collins
  20. Peter J. Cullen
  21. Yohei Yamauchi

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Abstract

Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor; however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics.

Science , this issue p. 856 , p. 861 ; see also p. 765

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  1. ScreenIT

    SciScore for 10.1101/2020.06.05.134114: (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 AuthenticationAuthentication: The decisions were based on single-cell and its microenvironment’s morphology and intensity features33.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and reagents: The following antibodies were used in this study: mouse anti-β actin (Sigma-Aldrich, A1978, WB 1:2000), mouse anti-ACE2 (Proteintech, 66699-1-Ig, WB 1:1000), mouse anti-GFP (Roche, 11814460001, WB 1:2000), rabbit anti-mCherry (Abcam, ab167453, WB 1:2000), rabbit anti-NRP1 (Abcam, ab81321, WB 1:1000), rabbit anti-SARS-CoV-2 Spike RBD (S1 epitope) (Sino Biologicals, 40592-T62, WB 1:1000), rabbit anti-SARS nucleocapsid (N) polyclonal antibody (ROCKLAND, 200-401-A50, IFA 1:2000), mouse anti-SARS-CoV-2 Spike antibody [1A9] (S2 epitope) (GeneTex, GTX632604, WB 1:1000).
    anti-β actin
    suggested: (EarthOx Life Sciences Cat# E021020, RRID:AB_2572416)
    anti-ACE2
    suggested: None
    anti-GFP
    suggested: (T. Kaneko, Kyoto University; Kyoto; Japan Cat# anti-GFP_kyoto_univ, RRID:AB_2716624)
    anti-mCherry
    suggested: (Abcam Cat# ab167453, RRID:AB_2571870)
    anti-NRP1
    suggested: (Abcam Cat# ab81321, RRID:AB_1640739)
    anti-SARS nucleocapsid (N
    suggested: (Abcam Cat# ab81321, RRID:AB_1640739)
    anti-SARS-CoV-2
    suggested: None
    S2 epitope
    suggested: (Imported from the IEDB Cat# S2, RRID:AB_2833224)
    The cells were then washed with PBS before the addition of medium supplemented with anti-VSV-G I1 antibody (kindly provided by Markus Hoffmann, German Primate Center, Leibnitz).
    anti-VSV-G I1
    suggested: None
    After permeabilisation with 0.1% TritonX-100 in PBS, 1% BSA, the cells were blocked and stained in 1% BSA in PBS containing anti-SARS Nucleocapsid (N) rabbit polyclonal antibody or anti-SARS Spike (S) monoclonal antibody and further stained with Hoechst (1:10000) and appropriate Alexa Fluor (488/594/647)-conjugated secondary antibodies (Thermo Fisher Scientific).
    anti-SARS
    suggested: None
    Secondary antibody AlexaFluor 546 goat anti-mouse IgG (Invitrogen Molecular Probes, Cat. No. A11003) was added to the cells (4μg/mL in diluted blocking buffer).
    anti-mouse IgG
    suggested: (Thermo Fisher Scientific Cat# A-11003, RRID:AB_2534071)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and transfection: Calu-3, Caco-2 (a kind gift from Dr Darryl Hill), HeLa, HEK293T and Vero E6 cell lines were originally sourced from the American Type Culture Collection.
    Caco-2
    suggested: None
    Cells were grown in DMEM medium (Sigma-Aldrich) supplemented with 10% (vol/vol) FCS (Sigma-Aldrich) and penicillin/streptomycin (Gibco) with the exception of Calu-3 cells that were grown in Eagle’s minimal essential medium (MEM+GlutaMAX; Gibco™, ThermoFischer) supplemented with 10% FCS 0.1mM non-essential amino acids (NEAA), 1mM sodium pyruvate, 100 IU/ml streptomycin and 100 μg/ml penicillin.
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    PPC-1 human primary prostate cancer cells were obtained from Erkki Ruoslahti laboratory at Cancer Research Center, Sanford-Burnham-Prebys Medical Discovery Institute.
    PPC-1
    suggested: ATCC Cat# HTB-190, RRID:CVCL_4778)
    M21 human melanoma cells were obtained from David Cheresh at University of California San Diego.
    M21
    suggested: RRID:CVCL_D031)
    The culture supernatant was passaged twice more on Vero E6 cells until cytopathic effect was observed and then once on Caco-2 cells to produce the stock used in the experiments.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Lentiviral particles were produced and harvested in HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    HeLa cells were transduced with lentiviral particles to produce stably expressing cell lines.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Software and Algorithms
    SentencesResources
    SARS-CoV-2 isolation and infection: A clinical specimen in viral transport medium, confirmed SARS-CoV-2 positive by qRT-PCR (kindly proved by Dr Lance Turtle, University of Liverpool), was adjusted to 2 ml with OptiMEM (Gibco™, ThermoFisher), filtered through a 0.2 μm filter and used to infect Vero E6 cells.
    ThermoFisher
    suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)

    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 10.1126/science.abd3072
  3. ScreenIT

    SciScore for 10.1101/2020.06.05.134114: (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 AuthenticationThe decisions were based on singlecell and its microenvironment’s morphology and intensity features33 .

    Table 2: Resources

    Antibodies
    SentencesResources
    Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism.
    ACE2
    suggested: None
    Following a GFP-nanotrap , the isolates were probed with an antibody raised against S1 .
    S1
    suggested: None
    All three monoclonal antibodies bound to the NRP1 b1b2 domain but only mAb#3 bound to the CendR-binding pocket ( Figure 3E , Extended Figure 3A) , as defined by a reduced ability to bind to a b1b2 mutant that targets residues ( S346 , E348 , T349 ) at the opening of the binding pocket12 ( Figure 2C) .
    S346
    suggested: (Abcam Cat# ab62464, AB_942199)
    The interaction between S1 and NRP1 can be reduced by monoclonal antibodies that bind to the CendR-binding pocket of NRP1 b1 .
    NRP1
    suggested: None
    MATERIALS AND METHODS Antibodies and reagents The following antibodies were used in this study: mouse anti-β actin ( Sigma-Aldrich , A1978 , WB 1:2000) , mouse anti-ACE2 ( Proteintech , 66699-1-Ig , WB 1:1000) , mouse anti-GFP ( Roche , 11814460001 , WB 1:2000) , rabbit anti-mCherry ( Abcam , ab167453 , WB 1:2000) , rabbit anti-NRP1 ( Abcam , ab81321 , WB 1:1000) , rabbit antiSARS-CoV-2 Spike RBD ( S1 epitope ) ( Sino Biologicals , 40592-T62 , WB 1:1000) , rabbit anti-SARS nucleocapsid ( N ) polyclonal antibody ( ROCKLAND , 200-401-A50 , IFA 1:2000) , mouse anti-SARS-CoV-2 Spike antibody [ 1A9 ] ( S2 epitope ) ( GeneTex , GTX632604 , WB 1:1000)
    anti-β actin
    suggested: (EarthOx Life Sciences Cat# E021020, AB_2572416)
          <div style="margin-bottom:8px">
        <div><b>anti-GFP</b></div>
        <div>suggested: (T. Kaneko, Kyoto University; Kyoto; Japan Cat# anti-GFP_kyoto_univ, <a href="https://scicrunch.org/resources/Any/search?q=AB_2716624">AB_2716624</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-mCherry</b></div>
        <div>suggested: (Abcam Cat# ab167453, <a href="https://scicrunch.org/resources/Any/search?q=AB_2571870">AB_2571870</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>antiSARS-CoV-2</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-SARS nucleocapsid ( N</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-SARS-CoV-2</b></div>
        <div>suggested: (Abcam Cat# ab272854, <a href="https://scicrunch.org/resources/Any/search?q=AB_2847844">AB_2847844</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>S2 epitope </b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were then washed with PBS before the addition of medium supplemented with anti-VSV-G I1 antibody ( kindly provided by Markus Hoffmann , German Primate Center , Leibnitz) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-VSV-G I1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After permeabilisation with 0.1 % TritonX-100 in PBS , 1 % BSA , the cells were blocked and stained in 1 % BSA in PBS containing anti-SARS Nucleocapsid ( N ) rabbit polyclonal antibody or anti-SARS Spike ( S ) monoclonal antibody and further stained with Hoechst ( 1:10000 ) and appropriate Alexa Fluor ( 488/594/647)conjugated secondary antibodies ( Thermo Fisher Scientific) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-SARS</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibody AlexaFluor 546 goat anti-mouse IgG ( Invitrogen Molecular Probes , Cat. No. A11003 ) was added to the cells ( 4µg/mL in diluted blocking buffer) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-mouse IgG</b></div>
        <div>suggested: (Thermo Fisher Scientific Cat# A-11003, <a href="https://scicrunch.org/resources/Any/search?q=AB_2534071">AB_2534071</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The percentage of infected cells was 73 % lower in the HeLaNRP1KO+ACE2 cells compared to HeLaWT+ACE2 cells ( Figure 1G) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLaWT+ACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To test the functional relevance of this association , we transiently expressed GFP , NRP1-GFP or NRP1 ( T316R)-GFP in HeLaNRP1KO+ACE2 cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLaNRP1KO+ACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using mAb#3 and a control monoclonal antibody targeting avian influenza A virus ( H11N3 ) hemagglutinin we found that mAb#3 inhibited SARS-CoV-2 infection of Caco-2 and Calu-3 cells by 38 % ( Figure 3F) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Calu-3</b></div>
        <div>suggested: BCRJ Cat# 0264, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0609">CVCL_0609</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and transfection Calu-3 , Caco-2 ( a kind gift from Dr Darryl Hill) , HeLa , HEK293T and Vero E6 cell lines were originally sourced from the American Type Culture Collection .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Caco-2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PPC-1 human primary prostate cancer cells were obtained from Erkki Ruoslahti laboratory at Cancer Research Center ,</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PPC-1</b></div>
        <div>suggested: ATCC Cat# HTB-190, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_4778">CVCL_4778</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate a NRP1-null HeLa cell line , the following guide RNA was cloned into pSpCas9 ( BB)-2A-Puro ( PX459): 5’-GATCGACGTTAGCTCCAACG- 3’ .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLa</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 isolation and infection A clinical specimen in viral transport medium , confirmed SARS-CoV-2 positive by qRT-PCR ( kindly proved by Dr Lance Turtle , University of Liverpool) , was adjusted to 2 ml with OptiMEM ( Gibco™ , ThermoFisher) , filtered through a 0.2 µm filter and used to infect Vero E6 cells .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell immunostaining and confocal microscopy PPC-1 and M21 cells were seeded in a 24-well plate ( 50,000 cells/well ) with coverslips and allowed to grow until the next day .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>M21</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the GFP- and mCherry-based immunoprecipitations , HEK293T cells were transfected with GFP or mCherry constructs using polyethylenimine ( Sigma-Aldrich) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
      </div>
    </td></tr></table>

    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 OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.06.05.134114 on bioRxiv