A Newly Engineered A549 Cell Line Expressing ACE2 and TMPRSS2 Is Highly Permissive to SARS-CoV-2, Including the Delta and Omicron Variants

  1. Ching-Wen Chang
  2. Krishna Parsi
  3. Mohan Somasundaran
  4. Emma Vanderleeden
  5. Ping Liu
  6. John Cruz
  7. Alyssa Cousineau
  8. Robert Finberg
  9. Evelyn Kurt-Jones

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Abstract

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses—underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 (“ACE2plus” and the subclone “ACE2plusC3”) are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

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  1. Version published to 10.3390/v14071369
  2. ScreenIT

    SciScore for 10.1101/2021.12.31.474593: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ACE2 and TMPRSS2 antibodies were purchased from R&D SYSTEMS (AF933) and Santa Cruz (sc-515727)
    ACE2
    suggested: None
    TMPRSS2
    suggested: (Santa Cruz Biotechnology Cat# sc-515727, RRID:AB_2892118)
    AF933
    suggested: (R and D Systems Cat# AF933, RRID:AB_355722)
    To improve the infectivity, the cell populations with a higher ACE2 expression in clone 43.20 were FACS sorted using ACE2-specific antibody (AF933, R&D Systems).
    ACE2-specific
    suggested: None
    Fixed cells were either labeled with a human monoclonal antibody conjugated with Alexa-488 against the spike antigen (10.1371/journal.pmed.0030237) or a mouse monoclonal antibody that recognizes the NP antigen (SinoBiological, #40143-MM08) by incubation for 2 hours at 4 °C.
    Alexa-488 against the spike antigen (10.1371/journal.pmed.0030237)
    suggested: None
    After washing with saline-Tween 20 (0.05%), the cells were labeled with an anti-mouse goat secondary antibody conjugated with Alexa-594 by incubation for 1 h at 4 °C.
    anti-mouse goat
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549 cells (CCL-185), Calu-3 (HTB-55) and Vero E6 (CRL-1586) were obtained from ATCC.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Commercial cell lines, A549ACE2 and A549ACE2/TMPRSS2 were purchased from InvivoGen.
    A549ACE2/TMPRSS2
    suggested: None
    Antiviral compounds Remdesivir and EIDD-193 were purchased from MedChemExpress
    EIDD-193
    suggested: None
    The test compounds were solubilized in DMSO to yield 10 mM stock solutions for cell culture studies. 2.2. Generation of A54943.20, A549ACE2plus, A549ACE2plusC3 cells: A549 cells were first transduced with human ACE2-expressing lentivirus (~2 × 105 CFU/ml) and selected with 1ug/ml of puromycin as previous described (Koupenova et al., 2021).
    A549ACE2plusC3
    suggested: None
    A549
    suggested: None
    5 Vero E6 cells were seeded to each well of 12-well plates and cultured at 37 °C, 5% CO2 for 18 h.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    The generation of pseudotyped lentiviral particles was based on the protocol as previous described (Crawford et al., 2020). pcDNA3.3-WA1-SARS2, pcDNA3.3-Alpha-SARS2, pcDNA3.3-Beta-SARS2, pcDNA3.3-Delta-SARS2 were gifts from David Nemazee
    pcDNA3.3-WA1-SARS2
    suggested: None
    pcDNA3.3-Alpha-SARS2
    suggested: None
    pcDNA3.3-Beta-SARS2
    suggested: None
    pcDNA3.3-Delta-SARS2
    suggested: None
    Addgene plasmid #170442, #170451, #170449, 172320). pcDNA3.1-Omicron-SARS2 (# MC0101274) was purchased from GenScript.
    pcDNA3.1-Omicron-SARS2
    suggested: None
    pHAGE-EF1a-ACE2PGK-puroR (internal ID 55490) and pHAGE-EF1a-TMPRSS2-PGK-puroR (internal ID 11816) plasmids were from hOrfeome 5.1 collection in the Maehr lab.
    pHAGE-EF1a-ACE2PGK-puroR
    suggested: None
    pHAGE-EF1a-TMPRSS2-PGK-puroR
    suggested: None
    Software and Algorithms
    SentencesResources
    The images were processed using MetaXpress Software.
    MetaXpress
    suggested: (MetaXpress, RRID:SCR_016654)
    All tests were performed using Prism 9 (GraphPad Software).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 18. 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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  3. Version published to 10.1101/2021.12.31.474593v1 on bioRxiv