1. Microbiology and Infectious Disease

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

  1. Mart M Lamers
  2. Anna Z Mykytyn
  3. Tim I Breugem
  4. Yiquan Wang
  5. Douglas C Wu
  6. Samra Riesebosch
  7. Petra B van den Doel
  8. Debby Schipper
  9. Theo Bestebroer
  10. Nicholas C Wu
  11. Bart L Haagmans  Is a corresponding author
  1. Erasmus MC, Netherlands
  2. University of Illinois at Urbana-Champaign, United States
  3. Invitae Corporation, United States
https://doi.org/10.7554/eLife.66815
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  1. Mart M Lamers
  2. Anna Z Mykytyn
  3. Tim I Breugem
  4. Yiquan Wang
  5. Douglas C Wu
  6. Samra Riesebosch
  7. Petra B van den Doel
  8. Debby Schipper
  9. Theo Bestebroer
  10. Nicholas C Wu
  11. Bart L Haagmans
(2021)
Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation
eLife 10:e66815.
https://doi.org/10.7554/eLife.66815
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Abstract

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.

Data availability

All scripts used for data processing are deposited in GitHub: https://github.com/wchnicholas/SARS_CoV2_mutation. Raw sequencing data are available at the NIH Short Read Archive under accession number: BioProject PRJNA694097.

The following data sets were generated

Article and author information

Author details

  1. 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

  2. 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

  3. Tim I Breugem

    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:" 0000-0002-5558-7043

  4. Yiquan Wang

    Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1954-9808

  5. Douglas C Wu

    Invitae Corporation, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6179-3110

  6. Samra Riesebosch

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

  7. Petra B van den Doel

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

  8. Debby Schipper

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

  9. Theo Bestebroer

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

  10. Nicholas C Wu

    Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. 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

Netherlands Organization for Health Research and Development (10150062010008)

  • Bart L Haagmans

PPP allowance (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 College London, United Kingdom

Version history

  1. Received: January 22, 2021
  2. Accepted: April 8, 2021
  3. Accepted Manuscript published: April 9, 2021 (version 1)
  4. Version of Record published: May 18, 2021 (version 2)

Copyright

© 2021, Lamers 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. Mart M Lamers
  2. Anna Z Mykytyn
  3. Tim I Breugem
  4. Yiquan Wang
  5. Douglas C Wu
  6. Samra Riesebosch
  7. Petra B van den Doel
  8. Debby Schipper
  9. Theo Bestebroer
  10. Nicholas C Wu
  11. Bart L Haagmans
(2021)
Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation
eLife 10:e66815.
https://doi.org/10.7554/eLife.66815

Share this article

https://doi.org/10.7554/eLife.66815

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Further reading

    1. Microbiology and Infectious Disease

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

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    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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