A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2

  1. Yang Zhang
  2. Wuhui Song
  3. Shuiye Chen
  4. Zhenghong Yuan
  5. Zhigang Yi

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Abstract

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

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  3. ScreenIT

    SciScore for 10.1101/2020.09.11.294330: (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
    Antibodies: Anti-β-actin antibody (Sigma; A1978) was used at 1:5000 dilution; Anti-HA antibody (CST; 37243) was used at 1:000 dilution; Anti-GFP antibody (Santa Cruz;sc-9996) was used at 1:1000 dilution; Anti-ZAPL antibody (Proteintech; 16820-1-AP) was used at 1:1000 dilution; Anti-N antibody(GeneTex; GTX632269) was used at 1:500 dilution; Goat-anti-mouse IRDye 800CW secondary antibody (licor; 926-32210) was used at 1:10,000 dilution.
    Anti-β-actin
    suggested: None
    Anti-HA
    suggested: (Rockland Cat# 600-431-384, RRID:AB_218003)
    Anti-GFP
    suggested: (Santa Cruz Biotechnology Cat# sc-9996, RRID:AB_627695)
    Anti-ZAPL
    suggested: None
    Anti-N antibody(GeneTex; GTX632269
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: The human hepatoma cells Huh 7, baby hamster kidney cells BKH-21, Vero E6 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (www.cellbank.org.cn) and routinely maintained in Dulbecco’s modified medium supplemented with 10 % FBS (Gibco) and 25 mM HEPES (Gibco).
    Vero E6
    suggested: RRID:CVCL_XD71)
    Huh7-GFP, Huh7-N, Huh7-ZAPL cell line was routinely maintained in the medium supplemented with 0.5 μg/ml blasticidin.
    Huh7-ZAPL
    suggested: None
    Lentivirus pseudoparticle: VSV-G-pseudotyped lentiviral particles were prepared by co-transfection of VSV-G, HIV gag-pol and lentiviral provirus plasmids into HEK293T cells.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Experimental Models: Organisms/Strains
    SentencesResources
    Huh7-GFP, Huh7-N, Huh7-ZAPL cell line was routinely maintained in the medium supplemented with 0.5 μg/ml blasticidin.
    Huh7-GFP
    suggested: None

    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.

    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.

  4. Version published to 10.1101/2020.09.11.294330 on bioRxiv