Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition

  1. Xi He
  2. Shuo Quan
  3. Min Xu
  4. Silveria Rodriguez
  5. Shih Lin Goh
  6. Jiajie Wei
  7. Arthur Fridman
  8. Kenneth A. Koeplinger
  9. Steve S. Carroll
  10. Jay A. Grobler
  11. Amy S. Espeseth
  12. David B. Olsen
  13. Daria J. Hazuda
  14. Dai Wang

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Abstract

SARS-CoV-2 research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a non-infectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlights the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.12.422532: (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

    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: Human embryonic kidney 293T (Clontech, 632180), human lung carcinoma A549 (ATCC, CCL-185) and human hepatoma Huh7.5 (Apath) cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen,
    A549
    suggested: None
    10-013-CV); African green monkey kidney Vero (ATCC, CCL-81) and human lung epidermoid carcinoma Calu-1 (ATCC, HTB-54) cells were cultured in Eagle’s Minimum Essential Medium (ATCC, 30-2003) and McCoy’s 5a medium Modified (ATCC, 30-2007), respectively.
    Calu-1
    suggested: ATCC Cat# HTB-54, RRID:CVCL_0608)
    Cryopreservation of electroporated cells: The 293T cells were prepared and electroporated as described above.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    1 were re-graphed in Prism (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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 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.
    • Thank you for including a protocol registration statement.

    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.

  2. Version published to 10.1101/2020.12.12.422532v1 on bioRxiv