Screening of SARS-CoV-2 antivirals through a cell-based RNA-dependent RNA polymerase (RdRp) reporter assay

  1. Timsy Uppal
  2. Kai Tuffo
  3. Svetlana Khaiboullina
  4. Sivani Reganti
  5. Mark Pandori
  6. Subhash C. Verma

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Abstract

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  1. Version published to 10.1016/j.cellin.2022.100046
  2. ScreenIT

    SciScore for 10.1101/2022.04.04.486994: (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 mouse anti-Flag (M2, Sigma-Aldrich), and mouse anti-GAPDH (G8140, US Biological, Salem MA) antibodies were used in this study.
    anti-GAPDH
    suggested: (US Biological Cat# G8140-01L, RRID:AB_2278712)
    After 3 weeks of selection, expanded clones were examined for the expression of Flag-tagged nsp12 (RdRp) by western blot using anti-Flag antibody.
    anti-Flag
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Establishment of the Stable cell lines: Permissive HEK293 cells were transfected with RdRp-luciferase reporter and pcDNA3.1.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    After 24h, cells were selected using 250ug/mL G418 (for 293/RdRp cell lines) or 250ug/mL G418 and 25ug/mL hygromycin (for 293/RdRp-nsp5,7,8,12-Flag cell lines).
    293/RdRp
    suggested: None
    293/RdRp-nsp5,7,8,12-Flag
    suggested: None
    For relative viral genome quantification post infection and compound treatment, control or USA-WA1/2020 virus was added onto the Vero E6 cells plated in a 24-well plate (100,000 cells per well) for 2h (37 °C and 5% CO2).
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    Establishment of the Stable cell lines: Permissive HEK293 cells were transfected with RdRp-luciferase reporter and pcDNA3.1.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    The input samples and the IP samples were resuspended in 45μL of SDS-PAGE loading buffer and denatured at 95°C for 5 min, resolved by SDS-PAGE and western blotted onto a 0.45 μM nitrocellulose membrane using standard protocols (Bio-Rad Laboratories, Hercules, CA, United States).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    The absolute half-maximal inhibitory concentrations (IC50) of dasabuvir for SARS-CoV-2 USA-WA1/2020 and B.1.617.2 were calculated, based on the amounts of residual virus in the supernatant at each concentration, using the Graphpad Prism™. 2.8. Plaque Assay: Vero E6 cells (100,000 cells/well) in 1mL medium were seeded in a 24-well culture plate for 2h (37 °C and 5% CO2).
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)
    Statistical analyses were performed using Prism 8.0 software (GraphPad Inc.).
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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

    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 10.1101/2022.04.04.486994 on bioRxiv