Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening

  1. Tomohisa Tanaka
  2. Akatsuki Saito
  3. Tatsuya Suzuki
  4. Yoichi Miyamoto
  5. Kazuo Takayama
  6. Toru Okamoto
  7. Kohji Moriishi

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Abstract

No abstract available

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  1. Version published to 10.1016/j.antiviral.2022.105268
  2. ScreenIT

    SciScore for 10.1101/2021.12.23.474055: (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 following antibodies were used in this study: mouse anti-SARS-CoV-2 nucleocapsid (N) monoclonal antibody (3A9; Cell Engineering Co. Osaka, Japan), mouse anti-SARS-CoV-2 nsp8 antibody (5A10; GeneTex, Irvine, CA), mouse anti-β-actin antibody (AC-74; Sigma–Aldrich), HRP-conjugated rabbit anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Alexa 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific, Waltham, MA).
    anti-SARS-CoV-2 nucleocapsid (N
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    anti-β-actin
    suggested: (Sigma-Aldrich Cat# A2228, RRID:AB_476697)
    anti-mouse IgG
    suggested: None
    The cells were then incubated with a dilution buffer (PBS with 1% BSA and 0.1% saponin) containing anti-SARS-CoV-2 N antibody (1:500) at room temperature for 90 min.
    anti-SARS-CoV-2 N
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The human embryonal kidney 293T, human hepatoma Huh7 and African green monkey kidney VeroE6 cell lines were cultured in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Gibco, Cambrex, MD) and 1% sodium pyruvate (Sigma–Aldrich), 1% nonessential amino acid (Sigma–Aldrich) and 1% penicillin–streptomycin solutions (Nacalai Tesque, Kyoto, Japan).
    Huh7
    suggested: None
    G418 (Nacalai Tesque) was further added to the culture medium at a final concentration of 1 mg/ml for the establishment and maintenance of the VeroE6/Rep cell line.
    VeroE6/Rep
    suggested: None
    Recombinant DNA
    SentencesResources
    Method details: Cloning, preparation and transfection of the BAC vector: Overviews of the design and construction of the replicon-BAC vector (pBAC-SCoV2-Rep-Reo) are shown in Figs.
    pBAC-SCoV2-Rep-Reo
    suggested: None
    First, we generated a pBAC cassette vector that encodes the cytomegalovirus (CMV) immediate-early promoter (CMVp) immediately upstream of the 5’ UTR of SARS-CoV-2 and the N gene followed by the 3’ UTR, synthetic 25-nt poly(A), the hepatitis delta virus ribozyme and BGH polyadenylation sequences, as the PCR template for fragment F10 (Fig.
    pBAC
    suggested: None
    Fragment F9, which is composed of, in order, a spacer (AceI site), the 3’-end portion of ORF8, TRS-B, the Reo gene, a spacer (BamHI site), the 3’-end portion of ORF8, and TRS-B, was subcloned into pCR-blunt (Fig. 1A, S1A).
    pCR-blunt
    suggested: RRID:Addgene_112638)
    To construct the pBAC cassette, the CMVp and the 5’-terminal region of SARS-CoV-2 (nt 1-683) were amplified by PCR from pcDNA3.1 and viral cDNA, respectively, combined by overlap PCR, and then cloned into pSMART-BAC v2.0 (Lucigen Corp., Middletown, WI).
    pSMART-BAC v2.0
    suggested: None
    Subsequently, the 3’-terminal region of SARS-CoV-2 (nt 28,134-29,902) and polyadenylation sequence were amplified by PCR from the viral cDNA and pcDNA3.1, respectively, connected across the synthesized sequence consisting of 25-nt poly(A) and the hepatitis delta virus ribozyme (Almazan et al., 2006) by overlap PCR, and cloned into the BAC described above.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    DNA preparation was carried out using NucleoSpin (Takara Bio) and NucleoBond Xtra BAC Kits (Takara Bio) for small-scale and large-scale replicon-BAC vector preparation, respectively.
    replicon-BAC
    suggested: None
    The N region was amplified with the primers listed in Table 1 and cloned into pBluescriptII.
    pBluescriptII
    suggested: None
    Software and Algorithms
    SentencesResources
    The cells were analyzed with BD FACSCalibur and CellQuest software (BD Biosciences).
    BD FACSCalibur
    suggested: (BD FACSCalibur Flow Cytometry System, RRID:SCR_000401)

    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 10.1101/2021.12.23.474055 on bioRxiv