The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus entry inhibitor

  1. Emmanuelle V. LeBlanc
  2. Che C. Colpitts

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Abstract

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective antiviral countermeasures are urgently needed as vaccine development and deployment against an emerging coronavirus takes time, even in the best-case scenario. The green tea catechin, epigallocatechin gallate (EGCG), has broad spectrum antiviral activity. We demonstrate here that EGCG prevents murine and human coronavirus infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from highly pathogenic coronaviruses, as well as a bat coronavirus poised to emerge into humans. We show that EGCG treatment reduces coronavirus attachment to target cell surfaces. Our results demonstrate the potential for the development of pan-coronavirus attachment inhibitors to protect against current and future emerging coronaviruses.

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  1. ScreenIT

    SciScore for 10.1101/2021.06.21.449320: (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
    The bulk population was single-cell cloned by limiting dilution, and ACE2 expression of clonal populations was determined by Western blotting using a rabbit monoclonal ACE2 antibody (Invitrogen MA5-32307), mouse monoclonal actin (Abcam ab8226) or tubulin (Abcam ab7291) antibodies, and a LICOR Odyssey CLx with appropriate secondary antibodies.
    ACE2
    suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589)
    actin
    suggested: (Abcam Cat# ab8226, RRID:AB_306371)
    or tubulin
    suggested: (Abcam Cat# ab8226, RRID:AB_306371)
    Cells were incubated with primary mouse IgG anti-coronavirus group antibody MAB9013 (Millipore Sigma; diluted 1:500) for 1 h at room temperature, followed by addition of secondary Alexa Fluor 488 anti-mouse IgG Fab 2 antibody (Cell Signaling Technology 4408S) for 1 h at room temperature.
    anti-coronavirus group
    suggested: (Millipore Cat# MAB9013, RRID:AB_95425)
    anti-mouse IgG Fab 2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: HEK293T/17 (ATCC CRL-11268), L929 (ATCC CCL-1), Huh7 (JCRB0403), HCT-8 (ATCC CCL-244) and 17CL1 (BEI Resources NR-53719) cells were cultured in Dulbecco’s minimal essential medium (DMEM) with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin at 37°C in 5% CO2.
    L929
    suggested: ECACC Cat# 86032004, RRID:CVCL_4238)
    For HCoV-OC43, HCT-8 cells were infected (MOI of 0.01) and incubated at 33°C in 5% CO2 until full CPE were observed (∼5 days post-infection).
    HCT-8
    suggested: None
    Infectivity assays: Huh7 cells (1.6 × 106 cells/well in 6-well plates) were infected with approximately 100 PFU of HCoV-229E pre-exposed for 10 min at 37°C to EGCG or dimethyl sulfoxide (DMSO) vehicle in DMEM.
    Huh7
    suggested: None
    Huh7 and A549 cells (4 × 105 cells/well in 24-well plates) were infected with approximately 200 focus-forming units (FFU) of HCoV-OC43, pre-exposed to EGCG or DMSO vehicle for 10 min at 37°C in DMEM.
    A549
    suggested: None
    VSV-SARS-CoV-2 S virions pre-exposed to EGCG or the DMSO vehicle control for 10 min at 37°C in DMEM were used to infect Huh7, A549-ACE2 or Calu-3 cell monolayers in duplicate wells of a 96-well plate.
    Calu-3
    suggested: None
    Pseudoparticle entry assays: Lentiviral pseudoparticles were generated in HEK293T/17 cells by co-transfection using Lipofectamine 2000 (Invitrogen 11668-019) (Crawford et al., 2020).
    HEK293T/17
    suggested: None
    Pseudoparticles were incubated for 10 min at 37°C with serially diluted EGCG and used to infect Huh7 or A549-ACE2 cells in triplicate in 96-well plates.
    A549-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    The lentiviral vector expressing human ACE2 (Dr. Sonja Best, Addgene 154981) was used with the lentiviral packaging plasmid psPAX2 (Dr. Didier Trono, Addgene 12260) and pCMV-VSV-G (Dr. Bob Weinberg, Addgene 8454). 2.3.
    psPAX2
    suggested: RRID:Addgene_12260)
    pCMV-VSV-G
    suggested: RRID:Addgene_8454)
    Software and Algorithms
    SentencesResources
    GFP signals were captured by Nikon Eclipse Ts2, processed using the Python Imaging Library to find the mean grey value, and plotted as percentage of inhibition.
    Python
    suggested: (IPython, RRID:SCR_001658)
    IC50 were calculated by nonlinear regression analysis using GraphPad Prism (version 9.0; GraphPad Software, Inc.). 2.6.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: 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.

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

    About SciScore

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  2. Version published to 10.1101/2021.06.21.449320v2 on bioRxiv
  3. Version published to 10.1101/2021.06.21.449320v1 on bioRxiv