Screening and testing for a suitable untransfected cell line for SARS-CoV-2 studies

  1. Claudia Pommerenke
  2. Ulfert Rand
  3. Cord C. Uphoff
  4. Stefan Nagel
  5. Margarete Zaborski
  6. Vivian Hauer
  7. Maren Kaufmann
  8. Corinna Meyer
  9. Sabine A. Denkmann
  10. Peggy Riese
  11. Kathrin Eschke
  12. Yeonsu Kim
  13. Zeljka Macak Safranko
  14. Ivan-Christian Kurolt
  15. Alemka Markotic
  16. Linda Brunotte
  17. Stephan Ludwig
  18. Luka Cicin-Sain
  19. Laura Steenpaß

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Abstract

At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus.

Author summary

In the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.09.195040: (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 variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Western blots: Antibodies against ACE2 (ab15348), TMPRSS2 (ab109131), and GAPDH (ab8245) were obtained from Abcam (Cambridge, UK).
    ACE2
    suggested: None
    TMPRSS2
    suggested: (Abcam Cat# ab109131, RRID:AB_10863728)
    GAPDH
    suggested: (Abcam Cat# ab8245, RRID:AB_2107448)
    Primary antibody used to stain dsRNA was mouse anti-dsRNA, clone rJ2 (Merck Millipore, Cat. no. MABE1134); secondary antibody was goat anti-rabbit-Cy5 (Life Technologies, Cat. no. A10523).
    anti-dsRNA
    suggested: (Millipore Cat# MABE1134, RRID:AB_2819101)
    anti-rabbit-Cy5
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Supernatants of infected cells were taken 5 dpi, serially diluted, and used to infect confluent Vero E6 cells on 96-well cell culture plates for one hour.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Briefly, alignment files were downloaded from CGHub via genetorrent, sorted (samtools 0.1.19) and converted to fastq files (bedtools v2.21.0).
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    bedtools
    suggested: (BEDTools, RRID:SCR_006646)
    STAR (2.5.3a) served as read mapper to the human genome Gencode 26, HTSeq (0.11.3) was used as count tool.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    HTSeq
    suggested: (HTSeq, RRID:SCR_005514)
    Raw counts were normalised and transformed to FPKM (fragments per kilobase million) via R/Bioconductor (3.6.9) loading DESeq2 (1.26.0) and visualised via ggplot2 (3.1.1).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Proteins on nitrocellulose membranes were visualized with the biotin/streptavidin-horseradish peroxidase system (GE Healthcare; Little Chalfont, UK) in combination with the “Renaissance Western Blot Chemoluminescence Reagent” (Perkin Elmer; Waltham, MA, USA).
    GE Healthcare
    suggested: (GE Healthcare, RRID:SCR_000004)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

  2. Version published to 10.1101/2020.07.09.195040v1 on bioRxiv