The use of Pseudotyped Coronaviruses for the Screening of Entry Inhibitors: Green Tea Extract Inhibits the Entry of SARS-CoV-1, MERSCoV, and SARS-CoV-2 by Blocking Receptor-spike Interaction

  1. Jeswin Joseph
  2. Thankamani Karthika
  3. Valiyathara Rajan Akshay Das
  4. Victor Stalin Raj

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Abstract

Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike protein to bind to a host cell surface receptor. The ongoing outbreak of SARS-CoV-2 inculcates the need for the development of effective intervention strategies.

Objectives:

In this study, we aim to produce pseudotyped coronaviruses of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 and show its applications, including virus entry, neutralization, and screening of entry inhibitors from natural products.

Methods:

Here, we generated VSV-based pseudotyped coronaviruses (CoV-PVs) for SARS-CoV-1, MERS-CoV, and SARS-CoV-2. Recombinant spike proteins of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 were transiently expressed in HEK293T cells followed by infection with recombinant VSV. High titer pseudoviruses were harvested and subjected to distinct validation assays, which confirms the proper spike pseudotyping. Further, specific receptor-mediated entry was confirmed by antibody neutralization and soluble form of receptor inhibition assay on Vero E6 cells. Next, these CoV-PVs were used for screening of antiviral activity of natural products such as green tea and Spirulina extract.

Results:

Here, we generated VSV-based pseudotyped coronaviruses (CoV-PVs) for SARS-CoV-1, MERS-CoV, and SARS-CoV-2. Recombinant spike proteins of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 were transiently expressed in HEK293T cells followed by infection with recombinant VSV. High titer pseudoviruses were harvested and subjected to distinct validation assays, which confirms the proper spike pseudotyping. Further, specific receptor-mediated entry was confirmed by antibody neutralization and soluble form of receptor inhibition assay on Vero E6 cells. Next, these CoV-PVs were used for screening of antiviral activity of natural products such as green tea and Spirulina extract.

Conclusion:

In summary, we demonstrated that pseudotyped viruses are an ideal tool for studying viral entry, quantification of neutralizing antibodies, and screening of entry inhibitors in a BSL-2 facility. Moreover, green tea might be a promising natural remedy against emerging coronaviruses.

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  1. Version published to 10.2174/1389201022666210810111716
  2. ScreenIT

    SciScore for 10.1101/2020.06.20.162701: (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
    , eluted with 200mM imidazole, and was analyzed by SDS PAGE and western blot using Goat anti-human ACE 2 polyclonal antibody (R&D, Cat.No-AF933)
    anti-human ACE
    suggested: None
    Surface expression was analyzed by incubation of either Goat anti-ACE2 or Goat anti-DPP4 (1:250) primary antibody and secondary antibody with rabbit anti-goat conjugated with Alexa Fluor 594 (Immunotag-ITIF59418).
    anti-ACE2
    suggested: None
    anti-DPP4
    suggested: None
    anti-goat
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Vero E6 and HEK293T cells were grown in DMEM (Lonza, 12-604F) supplemented with 10% FBS (MP, 29101) and 1% penicillin/streptomycin.
    HEK293T
    suggested: None
    BHK21 cells were maintained in EMEM (Lonza, 12-611F) supplemented with 10% FBS and 1% penicillin/streptomycin (Lonza,
    BHK21
    suggested: None
    Huh7 cells were grown in RPMI 1640 medium (Lonza, 12-115F) with 10% FCS and 1% penicillin/streptomycin.
    Huh7
    suggested: None
    For titration assay, a day before the experiment Vero E6 and Huh7 cells were seeded in a 96 well plate at 2×104 cells per well and 100μl of serially diluted (1:10) pseudoviruses were added to the cells and incubated for 1h.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    A synthetic construct of the full-length spike of SARS-CoV-2 (aa residues 1-1273) was commercially synthesized from Genewiz, UK.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    For all statistical analyses, the GraphPad Prism 5 was used.
    GraphPad Prism
    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 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:
    • 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.

  3. Version published to 10.1101/2020.06.20.162701v1 on bioRxiv