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

  • Curated by eLife

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    Evaluation Summary:

    This manuscript follows up on work documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2 for determining cell tropism and mode of cell entry. The paper describes a number of important findings: 1) That SARS-CoV-2 grown in Vero cells rapidly acquires MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations emerging that are not apparent in consensus sequence reads; 3) that factors such as fetal calf serum can influence the selection of mutant phenotypes, and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the propagation of SARS-CoV-2 to avoid adaptations to laboratory cell-lines/conditions and maintain the authenticity of clinical isolates. The work has relevance to other viruses and the use of permissive transformed cell lines.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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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.

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  1. Version published to 10.7554/elife.66815 on eLife
  2. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Lamers et al wanted to characterise the previously reported adaptation of SARS-CoV-2 to non-human (Vero) cells. Vero cells are commonly used by laboratories to grow experimental stocks of some viruses as these cells permit high titres of many viruses, they lack the ability to produce type I interferons (cytokines which could interfere with downstream assays), and their non-human nature means soluble factors in virus stocks are less likely to impact experiments in human cells. However, a number of reports have recently been published describing that growth of SARS-CoV-2 in Vero cells leads to loss of the SARS-CoV-2 Spike protein multibasic cleavage site (MBCS). This apparent adaptation to the Vero cell-line leads to a virus compromised in its ability to enter, and therefore replicate in, human cells, meaning that experimental results obtained in human cells using the Vero-adapted SARS-CoV-2 may not fully reflect the situation occurring with authentic SARS-CoV-2. It is therefore important for the research community to understand SARS-CoV-2 adaptation to laboratory cell-lines/conditions and to have propagation methods that are suitable for maintaining the authenticity of clinical virus isolates.

    The major finding of Lamers et al in this manuscript is that human cell-lines (e.g. Calu-3) and primary human organoid systems can be used to propagate clinical isolates of SARS-CoV-2 to high titres without the acquisition of 'laboratory adaptations'. To get to this finding, the authors carefully study the adaptation of a representative SARS-CoV-2 isolate in Vero cells, monitoring plaque size phenotypes and performing whole-genome deep sequencing to identify adaptive variants that appear in the viral Spike gene. These variants (including newly-described substitutions as well as deletions around the MBCS) are validated for their impact on viral infectivity in human and Vero cells using pseudovirus assays, fusion assays, and western blot assays, and their role in affecting the entry route of SARS-CoV-2 is dissected using pathway-specific inhibitors (such as camostat and E64D) and cell-lines with/without TMPRSS2 (an important protease for Spike cleavage). Importantly, using these assays and tools, the authors can make solid and well-reasoned arguments as to why SARS-CoV-2 adapts to Vero cells, and thus why certain culture conditions and cell substrates lead to a loss of SARS-CoV-2 genetic stability. Using similar tools, this also allows the authors to carefully study whether any adaptations occur when SARS-CoV-2 stocks are passaged in human cell substrates (such as Calu-3 or primary human organoids), and study culture conditions in Veros (such as expression of TMPRSS2) that prevent changes in SARS-CoV-2.

    The data in this manuscript are thorough and well-presented. Importantly, the conclusions are strongly supported by the data, particularly the overall take-home message that human cell substrates can be used to efficiently propagate SARS-CoV-2 isolates without introducing cell culture adaptations. However, beyond this simple message, the manuscript also provides new mechanistic insights into the reasons for such viral adaptations in the Vero cell system, and identifies previously undescribed adaptations in the MBCS region that will be valuable for other researchers to take note of. The authors also describe a methodological workflow to produce SARS-CoV-2 in human cells that highlights a buffer-exchange step to remove potentially interfering human cytokines/debris, and which will be useful for other researchers.

    Overall, the manuscript makes a clear and important contribution to the SARS-CoV-2 field and will be of interest to active researchers who are studying this virus experimentally.

  3. eLife

    Reviewer #1 (Public Review):

    This manuscript, which follows on from a recent eLife paper documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2, shows that growing SARS-CoV-2 on relevant epithelial cell lines or differentiated stem cell-derived culture systems prevents the emergence of MBCS mutations than impact on properties of S that contribute to cell tropism and the viral entry mechanism.

    The paper builds on the authors previous work and that of others, and in some respects the results are not surprising. Nevertheless, the paper sets out a number of important findings. 1) That SARS-CoV-2 grown in Vero cells rapidly acquire MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations that are not apparent in consensus sequence reads, 3) that factors such as the addition of fetal calf serum can influence the selection of mutant phenotypes and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the production of SARS-CoV-2, and potentially other viruses, avoiding the pitfalls that can arise from growing viruses in permissive transformed cell lines.

    The data and manuscript are clearly presented, and my concerns are minimal. Overall, the paper will make a useful addition to the SARS-CoV-2 literature and will be of value to researchers working not just of SARS-CoV-2 but on many other viruses.

  4. eLife

    Evaluation Summary:

    This manuscript follows up on work documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2 for determining cell tropism and mode of cell entry. The paper describes a number of important findings: 1) That SARS-CoV-2 grown in Vero cells rapidly acquires MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations emerging that are not apparent in consensus sequence reads; 3) that factors such as fetal calf serum can influence the selection of mutant phenotypes, and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the propagation of SARS-CoV-2 to avoid adaptations to laboratory cell-lines/conditions and maintain the authenticity of clinical isolates. The work has relevance to other viruses and the use of permissive transformed cell lines.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  5. ScreenIT

    SciScore for 10.1101/2021.01.22.427802: (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).
    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-conjugated secondary antibodies (1:400; Invitrogen) in blocking buffer for two hours at room temperature, washed two times with PBS, incubated for 10 minutes with Hoechst, washed twice with PBS, and mounted in Prolong Antifade (Invitrogen) mounting medium.
    Antifade ( Invitrogen ) mounting medium .
    suggested: None
    TMPRSS2 was stained using mouse-anti-TMPRSS2 (sc-515727, 1:200, Santa Cruz Biotechnology), and visualized with goat-anti-mouse (PO260, 1:100, Dako) horseradish peroxidase labeled secondary antibody, respectively.
    mouse-anti-TMPRSS2
    suggested: None
    sc-515727
    suggested: None
    ( PO260
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    VeroE6-TMPRSS2, VeroE6-GFP1-10, VeroE6-TMPRSS2-GFP1-10, and Calu-3-GFP1-10 cells were generated as described before (Mykytyn et al., 2021).
    Calu-3-GFP1-10
    suggested: None
    Next, virus preparations were aliquoted in 500 μl aliquots, stored at −80°C and thawed for titrations on VeroE6 cells.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Entry routes were determined by pre-treating monolayers of VeroE6 or VeroE6-TMPRSS2 cells with a concentration range of camostat mesylate (Sigma) or E64D (MedChemExpress) diluted in Opti-MEM I (1X) + GlutaMAX (Gibco) for 2 hours prior to infection with 1 × 103 pseudovirus.
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Transfected HEK-293T cells were incubated overnight at 37°C 5% CO2, resuspended in PBS and added to GFP1-10 expressing VeroE6, VeroE6-TMPRSS2 and Calu-3 cells in Opti-MEM I (1X) + GlutaMAX at a ratio of 1:80 (HEK-293T cells : GFP1-10 expressing cells).
    Calu-3
    suggested: None
    HEK-293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    VeroE6-TMPRSS2, VeroE6-GFP1-10, VeroE6-TMPRSS2-GFP1-10, and Calu-3-GFP1-10 cells were generated as described before (Mykytyn et al., 2021).
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    After a two-hour incubation, cells were washed three times with AdDF+++ to remove unbound particles.
    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)
    The obtained sequences were assembled and aligned using Benchling (
    Benchling
    suggested: (Benchling, RRID:SCR_013955)
    MAFFT algorithm).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Samples were imaged on a LSM700 confocal microscope using ZEN software (Zeiss).
    ZEN
    suggested: None
    Illumina sequencing: For deep-sequencing, RNA was extracted as described above and subsequently cDNA was generated using ProtoscriptII reverse transcriptase enzyme (New England BiotechnologieBioLabs) according to the manufacturer’s protocol.
    New England BiotechnologieBioLabs
    suggested: None
    The trimmed reads were aligned to the genome of Bavpat-1 with Bowtie2 (PMC3322381) using parameters: --no-discordant --dovetail --no-mixed --maxins 2000.
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    via: samtools mpileup --excl-flags 2048 --excl-flags 256 --fasta-ref (REFERENCE_FAASTA) --max-depth 50000 --min-MQ 30 --min-BQ 30 (BAM_FILE) | varscan pileup2cns --min-coverage 10 --min-reads2 2 --min-var-freq 0.01 --min-freq-for-hom 0.75 --p-value 0.05 --variants 1 > (snp_file)
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Plotting of mutation frequencies was done using R and ggplot2 (Hadley, 2016).
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Raw sequencing data will be submitted to the NIH Short Read Archive under accession number: BioProject PRJNA694097.
    Short Read Archive
    suggested: None
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Statistics: Statistical analysis was performed with the GraphPad Prism 8 and 9 software using an ANOVA or two-way ANOVA followed by a Bonferroni multiple-comparison test.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your code and data.

    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 45 and 46. 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

    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.

  6. Version published to 10.1101/2021.01.22.427802v1 on bioRxiv