1. Microbiology and Infectious Disease

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
https://doi.org/10.7554/eLife.64508
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Cite this article
  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
(2021)
SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site
eLife 10:e64508.
https://doi.org/10.7554/eLife.64508
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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.

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

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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.

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.

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  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
(2021)
SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site
eLife 10:e64508.
https://doi.org/10.7554/eLife.64508

Share this article

https://doi.org/10.7554/eLife.64508

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Further reading

    1. Microbiology and Infectious Disease

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

    Mart M Lamers, Anna Z Mykytyn ... Bart L Haagmans
    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.

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