A PCR amplicon-based SARS-CoV-2 replicon for antiviral evaluation

  1. Tomohiro Kotaki
  2. Xuping Xie
  3. Pei-Yong Shi
  4. Masanori Kameoka

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Abstract

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC 50 of remdesivir in this study was 0.29 μM, generally consistent to the IC 50 obtained using infectious SARS-CoV-2 in a previous study (0.77 μM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.

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  1. Version published to 10.1038/s41598-021-82055-0
  2. ScreenIT

    SciScore for 10.1101/2020.08.28.267567: (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
    After blocking with normal goat serum, the cells were incubated with primary mouse monoclonal antibodies (mAbs) (anti-N mAb [6H3: GeneTex] or anti-NSP8 mAb [5A10: GeneTex]) followed by a secondary antibody (goat anti-mouse IgG conjugated with Alexa Fluor 488).
    anti-N
    suggested: None
    anti-NSP8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK-293T cell (ATCC: CRL-3216) was maintained in the DMEM supplemented with 10% FBS.
    HEK-293T
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    The construction of a SARS-CoV-2 replicon DNA: The viral RNA extracted from the culture fluid of SARS-CoV-2–infected Vero E6 cell (provided by the National Institute of Infectious Diseases, Japan) was reverse transcribed into cDNA by the SuperScript III First Strand Synthesis system (Thermo Fisher Scientific) with random hexamer primers.
    Vero E6
    suggested: RRID:CVCL_XD71)
    The parameters for BHK-21 and CHO-K1 cells were as follows: voltage = 145 V; pulse length = 5 ms; pulse interval = 50 ms; number of pulses = 1; decay rate = 10%; polarity + as poring pulse and voltage = 20 V; pulse length = 50 ms; pulse interval = 50 ms; number of pulses = 5; decay rate = 40%; and polarity +/− as transfer pulse.
    BHK-21
    suggested: None
    The parameters for 293T cell was same as above except voltage 150 V and pulse length of 2.5 ms for poring pulse.
    293T
    suggested: None
    Antiviral treatment: The CHO-K1 cells electroporated with 5 μg of the replicon RNA were seeded as 1.5 × 104 cells/well in a 96-well plate.
    CHO-K1
    suggested: None
    Software and Algorithms
    SentencesResources
    The RNA was electrophoresed using DynaMarker RNA High for Easy Electrophoresis (BioDynamics Laboratory. Inc.) for the rough quality check.
    BioDynamics Laboratory
    suggested: None
    The IC50 and CC50 were calculated using a four-parameter logistic regression model from the GraphPad Prism 5 software (GraphPad Software Inc.).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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.
    • 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.

  3. Version published to 10.1101/2020.08.28.267567v1 on bioRxiv