SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

  1. Anna Z Mykytyn
  2. Tim I Breugem
  3. Samra Riesebosch
  4. Debby Schipper
  5. Petra B van den Doel
  6. Robbert J Rottier
  7. Mart M Lamers
  8. Bart L Haagmans

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Abstract

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.

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  1. Version published to 10.7554/elife.64508 on eLife
  2. ScreenIT

    SciScore for 10.1101/2020.09.07.286120: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512)
    Consent: For the tracheal aspirates, informed consent was obtained from parents and approval was given by the Medical Ethical Committee (METC no. MEC-2017-302).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were incubated with primary antibodies overnight at 4°C in blocking buffer, washed twice with PBS, incubated with corresponding secondary antibodies Alexa488-, 594 and 647-conjugated secondary antibodies (1:400; Invitrogen) in blocking buffer for 2 hours at room temperature, washed two times with PBS, incubated with indicated additional stains (TO-PRO3, phalloidin-633 (SC-363796, Santa Cruz Biotechnology), Hoechst), washed twice with PBS, and mounted in Prolong Antifade (Invitrogen) mounting medium.
    TO-PRO3 , phalloidin-633 ( SC-363796 , Santa Cruz Biotechnology) , Hoechst)
    suggested: None
    phalloidin-633
    suggested: None
    Antifade ( Invitrogen ) mounting medium .
    suggested: None
    ACE2 and TMPRSS2 were stained using goat-anti-hACE2 (AF933, 1:200, R&D Systems) and mouse-anti-TMPRSS2 (sc-515727, 1:200, Santa Cruz Biotechnology), and visualized with rabbit-anti-goat (P0160, 1:200, Dako) and goat-anti-mouse (PO260, 1:100, Dako) horseradish peroxidase labeled secondary antibody, respectively.
    ACE2
    suggested: None
    TMPRSS2
    suggested: None
    mouse-anti-TMPRSS2
    suggested: None
    sc-515727
    suggested: None
    rabbit-anti-goat ( P0160
    suggested: None
    ( PO260
    suggested: None
    Two hours post-infection, cells were washed three times with Opti-MEM I (1X) + GlutaMAX and replaced with medium containing anti-VSV-G neutralizing antibody (clone 8G5F11; Absolute Antibody) at a dilution of 1:50000 to block remaining VSV-G PPs.
    anti-VSV-G neutralizing antibody
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Calu-3 and Calu-3 stable cell lines were maintained in Eagle’s minimal essential medium (EMEM, ATCC®) supplemented with 20% FCS, penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.
    Calu-3
    suggested: None
    HEK-293T cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), sodium pyruvate (1mM,
    HEK-293T
    suggested: None
    SARS-CoV-2 (isolate BetaCoV/Munich/BavPat1/2020; European Virus Archive Global #026V-03883; kindly provided by Dr. C. Drosten) and SARS-CoV (isolate HKU39849) were propagated on Vero cells in Opti-MEM I (1X) + GlutaMAX (Gibco), supplemented with penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37°C in a humidified CO2 incubator.
    Vero
    suggested: None
    Aliquots of each dilution were added to monolayers of 2 × 104 VeroE6 cells in the same medium in a 96-well plate.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Entry route assay: VeroE6, Calu-3 and VeroE6-TMPRSS2 cells were seeded in 24 well plates and kept at 37°C 5% CO2 overnight to achieve 80-100% confluency by the next day.
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    After a 10 minute incubation step at 37°C 5% CO2, dispase was removed and cold 500 μL AdDF+++ was pipetted onto the apical side of the Transwell to dislodge the pseudostratified epithelial layer, which was subsequently mechanically sheared by pipetting using a P1000 tip.
    AdDF+++
    suggested: None
    Fusion events were quantified by detecting GFP+ pixels after 18 hours incubation at 37°C 5% CO2 using Amersham™
    Amersham™
    suggested: (Amersham Biosciences, RRID:SCR_013566)
    Data was analyzed using the ImageQuant TL 8.2 image analysis software (GE Healthcare) by calculating the sum of all GFP+ pixels per well.
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    For nuclear counting fluorescence microscopy images were obtained with a Carl ZEISS Vert.A1 microscope paired with an AxioCam ICm1 camera and Colibri 7 laser (469/38nm for GFP and 365/10nm for BFP) using ZEN analysis software (20x magnification)
    Colibri
    suggested: (Colibri, RRID:SCR_007606)
    Confocal microscopy images were taken on a LSM700 confocal microscope using ZEN software.
    ZEN
    suggested: None
    Statistical analysis: Statistical analysis was performed with the GraphPad Prism 5 and 8 software using a t-test, one way ANOVA or two-way ANOVA followed by a Bonferroni multiple-comparison test.
    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 26 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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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  3. Version published to 10.1101/2020.09.07.286120v1 on bioRxiv