1. Microbiology and Infectious Disease
Download icon

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  Is a corresponding author
  1. Erasmus MC, Netherlands
Research Article
  • Cited 27
  • Views 3,169
  • Annotations
Cite this article as: eLife 2021;10:e64508 doi: 10.7554/eLife.64508

Abstract

Coronavirus entry is mediated by the spike protein which 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 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.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files

Article and author information

Author details

  1. Anna Z Mykytyn

    Viroscience, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7188-6871

  2. Tim I Breugem

    Viroscience, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  3. Samra Riesebosch

    Viroscience, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  4. Debby Schipper

    Viroscience, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  5. Petra B van den Doel

    Viroscience, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.

  6. Robbert J Rottier

    Pediatric Surgery/Cell Biology, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9291-4971

  7. Mart M Lamers

    Viroscience, Erasmus MC, Rotterdam, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1431-4022

  8. Bart L Haagmans

    Viroscience Department, Erasmus MC, Rotterdam, Netherlands
    For correspondence
    b.haagmans@erasmusmc.nl
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6221-2015

Funding

Nederlandse Organisatie voor Wetenschappelijk Onderzoek (0.22.005.032)

  • Bart L Haagmans

ZonMw (10150062010008)

  • Bart L Haagmans

Health Holland (LSHM19136)

  • Bart L Haagmans

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Mark Marsh, University Coillege London, United Kingdom

Publication history

  1. Received: October 31, 2020
  2. Accepted: December 31, 2020
  3. Accepted Manuscript published: January 4, 2021 (version 1)
  4. Version of Record published: January 13, 2021 (version 2)

Copyright

© 2021, Mykytyn et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,169
    Page views
  • 532
    Downloads
  • 27
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Categories and tags

Research organism

Further reading

    1. Microbiology and Infectious Disease

    Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation

    Mart M Lamers et al.
    Research Advance Updated

    Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 – that expresses serine proteases – prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

    1. Epidemiology and Global Health
    2. Medicine
    3. Microbiology and Infectious Disease

    COVID-19: A Collection of Articles

    Edited by Diane M Harper et al.
    Collection

    eLife has published the following articles on SARS-CoV-2 and COVID-19.

    1. Epidemiology and Global Health
    2. Microbiology and Infectious Disease

    Efficacy of FFP3 respirators for prevention of SARS-CoV-2 infection in healthcare workers

    Mark Ferris et al.
    Research Advance Updated

    Background:

    Respiratory protective equipment recommended in the UK for healthcare workers (HCWs) caring for patients with COVID-19 comprises a fluid-resistant surgical mask (FRSM), except in the context of aerosol generating procedures (AGPs). We previously demonstrated frequent pauci- and asymptomatic severe acute respiratory syndrome coronavirus 2 infection HCWs during the first wave of the COVID-19 pandemic in the UK, using a comprehensive PCR-based HCW screening programme (Rivett et al., 2020; Jones et al., 2020).

    Methods:

    Here, we use observational data and mathematical modelling to analyse infection rates amongst HCWs working on ‘red’ (coronavirus disease 2019, COVID-19) and ‘green’ (non-COVID-19) wards during the second wave of the pandemic, before and after the substitution of filtering face piece 3 (FFP3) respirators for FRSMs.

    Results:

    Whilst using FRSMs, HCWs working on red wards faced an approximately 31-fold (and at least fivefold) increased risk of direct, ward-based infection. Conversely, after changing to FFP3 respirators, this risk was significantly reduced (52–100% protection).

    Conclusions:

    FFP3 respirators may therefore provide more effective protection than FRSMs for HCWs caring for patients with COVID-19, whether or not AGPs are undertaken.

    Funding:

    Wellcome Trust, Medical Research Council, Addenbrooke’s Charitable Trust, NIHR Cambridge Biomedical Research Centre, NHS Blood and Transfusion, UKRI.