SARS-CoV-2 variants reveal features critical for replication in primary human cells

  1. Marie O. Pohl
  2. Idoia Busnadiego
  3. Verena Kufner
  4. Irina Glas
  5. Umut Karakus
  6. Stefan Schmutz
  7. Maryam Zaheri
  8. Irene Abela
  9. Alexandra Trkola
  10. Michael Huber
  11. Silke Stertz
  12. Benjamin G. Hale

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Abstract

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.

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  1. Version published to 10.1371/journal.pbio.3001006
  2. Version published to 10.1101/2020.10.22.350207v3 on bioRxiv
  3. Version published to 10.1101/2020.10.22.350207v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.10.22.350207: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablePrimary human bronchial epithelial cells (BEpCs) from a 73 year-old female donor (donor 1) were purchased from Promocell (#C-12640).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    A mouse (#Ab00458-1.1) or rabbit (Ab00458-23.0) anti-dsRNA antibody (9D5; Lucerna-Chem) was used to stain for SARS-CoV-2 infected cells.
    #Ab00458-1.1
    suggested: None
    anti-dsRNA
    suggested: (Millipore Cat# MABE1134, RRID:AB_2819101)
    A mouse anti-β-tubulin IV antibody (#ab11315; Abcam), a mouse anti-MUC5AC antibody (#ab3649; Abcam), a rabbit anti-P63 antibody (#ab124762; Abcam), and a rat anti-uteroglobin antibody (#MAB4218; R&D Systems) was used to stain ciliated, goblet, basal, and club cells, respectively.
    anti-β-tubulin IV
    suggested: None
    anti-MUC5AC
    suggested: (Abcam Cat# ab3649, RRID:AB_2146844)
    anti-P63
    suggested: (Abcam Cat# 5353-1, RRID:AB_10898189)
    anti-uteroglobin
    suggested: (Abcam Cat# ab217304, RRID:AB_2756341)
    As secondary antibodies, anti-mouse IgG Alexa488 (#A-11029), anti-mouse IgG Alexa647 (#A-31571), anti-rabbit IgG Alexa546 (#A-10040), anti-rabbit IgG Alexa647 (#A-31573), and anti-rat IgG Alexa488 (#A-11006) antibodies were used (all from Thermo Fisher Scientific).
    anti-mouse IgG
    suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)
    anti-rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# A10040, RRID:AB_2534016)
    anti-rat IgG
    suggested: (Molecular Probes Cat# A-11006, RRID:AB_141373)
    #A-11006
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero-CCL81 cells (ATCC) and Vero-E6 cells (kindly provided by Volker Thiel, University of Bern, Switzerland) were cultured at 37°C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco), supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/mL of penicillin and 100 μg/mL of streptomycin (#15140-122; Gibco).
    Vero-CCL81
    suggested: None
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The sequences were aligned using MAFFT v7.271 [60] and the phylogeny was estimated using RAxML [61] with GTR+G+F model.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    RAxML
    suggested: (RAxML, RRID:SCR_006086)
    Plaque size quantification was performed using ImageJ analysis software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Filters were mounted using ProLong Gold Antifade Mountant (#P36930; Thermo Fisher Scientific) and z-stack images were acquired using a DMi8 microscope (Leica) and processed using the THUNDER Large Volume Computational Clearing algorithm (Leica).
    THUNDER
    suggested: (Thunder, RRID:SCR_016556)

    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: We did not find any issues relating to colormaps.

    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.

  5. Version published to 10.1101/2020.10.22.350207v1 on bioRxiv