Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 in vitro

  1. Mehmet Altay Unal
  2. Ceylan Verda Bitirim
  3. Gokce Yagmur Summak
  4. Sidar Bereketoglu
  5. Inci Cevher Zeytin
  6. Omur Besbinar
  7. Cansu Gurcan
  8. Dunya Aydos
  9. Ezgi Goksoy
  10. Ebru Kocakaya
  11. Zeynep Eran
  12. Merve Murat
  13. Nil Demir
  14. Zeynep Busra Aksoy Ozer
  15. Julia Somers
  16. Emek Demir
  17. Hasan Nazir
  18. Sibel Aysil Ozkan
  19. Aykut Ozkul
  20. Alpay Azap
  21. Acelya Yilmazer
  22. Kamil Can Akcali

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Abstract

Ribavirin is a guanosine analog with broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via in vitro, in silico, and molecular techniques. Ribavirin showed antiviral activity in Vero E6 cells following SARS-CoV-2 infection, whereas the drug itself did not show any toxic effect over the concentration range tested. In silico analysis suggested that ribavirin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, ribavirin was shown to decrease the expression of TMPRSS2 at both mRNA and protein levels 48 h after treatment. The suppressive effect of ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that ribavirin also showed an inhibitory effect on the TMPRSS2 enzyme. Based on these results, we hypothesized that ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effects of TMPRSS2 and ACE2 expression.

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  1. Version published to 10.1139/cjpp-2020-0734
  2. ScreenIT

    SciScore for 10.1101/2020.12.04.410092: (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
    Vero E6 and Caco2 cells were harvested with Trypsin-EDTA (2X) and single cell suspensions were stained using antibody against TMPRSS2 (Santa Cruze, sc-515727) after fixation (4% Paraformaldehyde, 15 min RT) and permeabilization (INTRA, 15 min RT) process.
    TMPRSS2
    suggested: None
    Monoclonal anti-ACE2 antibody (ProSci, Poway, CA,1:750diluted in 5% BSA blocking buffer) and goat anti-rabbit IgG-horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Dallas, TX, 1:2500 diluted in 5% BSA blocking buffer) were used as primary and secondary antibodies respectively for ACE2 expression.
    anti-ACE2
    suggested: None
    anti-rabbit IgG-horseradish peroxidase-conjugated antibody ( Santa Cruz Biotechnology , Dallas , TX
    suggested: None
    Monoclonal anti-β-actin antibody (SantaCruz,1:1500diluted in 5%BSA blocking buffer) and goat anti-mouse HRP conjugated (Pierce, Rockford, IL, antibody diluted in 5%BSA blocking buffer) were used as primary and secondary antibodies respectively for β-actin.
    anti-β-actin
    suggested: None
    anti-mouse HRP conjugated ( Pierce , Rockford , IL ,
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Human colonadeno carcinoma Caco-2 cells were maintained in DMEM high glucose supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, and 1% non-essential amino acids.
    Caco-2
    suggested: None
    Vero E6 and Caco-2 cells were plated on 96-well plates at a concentration of 10.000 cells/well and incubated 24 hours.
    Vero E6
    suggested: None
    Vero E6 and Caco2 cells were harvested with Trypsin-EDTA (2X) and single cell suspensions were stained using antibody against TMPRSS2 (Santa Cruze, sc-515727) after fixation (4% Paraformaldehyde, 15 min RT) and permeabilization (INTRA, 15 min RT) process.
    Caco2
    suggested: None
    Software and Algorithms
    SentencesResources
    All analysis was performed using Novo Express 1.3.0 Software (ACEA Biosciences,
    Novo Express
    suggested: (FCS Express, RRID:SCR_016431)

    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.
    • No funding statement was detected.
    • 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 10.1101/2020.12.04.410092v1 on bioRxiv