A SARS-CoV-2 mini-genome assay based on negative-sense RNA to study replication inhibitors and emerging mutations

  1. Thomas Vial
  2. Michael S. Oade
  3. Colin A. Russell
  4. Dirk Eggink
  5. Aartjan J.W. te Velthuis

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Abstract

Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus and the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Efforts to identify inhibitors of SARS-CoV-2 replication enzymes and better understand the mechanisms underlying viral RNA synthesis have largely relied on biosafety level 3 (BSL3) laboratories, limiting throughput and accessibility. Recently, replicon systems have been proposed that involve ~ 30 kb RNA-based replicons or large plasmids that express the viral structural and non-structural proteins (nsp) in addition to a positive-sense reporter RNA. Unfortunately, these assays are not user-friendly due to plasmid instability or a poor signal to background ratio. We here present a simple mini-genome assay consisting of a ~ 2.5 kb-long negative-sense, nanoluciferase-encoding sub-genomic reporter RNA that is expressed from a plasmid, and amplified and transcribed by the SARS-CoV-2 RNA polymerase core proteins nsp7, nsp8 and nsp12. We show that expression of nsp7, 8 and 12 is sufficient to obtain robust positive- and negative-sense RNA synthesis in cell culture, that addition of other nsps modulates expression levels, and that replication of the reporter RNA can be inhibited by active site mutations in nsp12 or the SARS-CoV-2 replication inhibitor remdesivir. The mini-genome assay provides a signal that is 170-fold above background on average, providing excellent sensitivity for high-throughput screens, while the use of small plasmids facilitates site-directed mutagenesis for fundamental analyses of SARS-CoV-2 RNA synthesis.

Importance statement

The impact of the COVID-19 pandemic has made it essential to better understand the basic biology of SARS-CoV-2, and to search for compounds that can block the activity of key SARS-CoV-2 replication enzymes. However, studies with live SARS-CoV-2 require biosafety level 3 facilities, while existing replicon systems depend on long positive-sense subgenomes that are often difficult to manipulate or produce a high background signal, limiting drug-screens and a rapid analysis of emerging SARS-CoV-2 mutations during the COVID-19 pandemic. To make it easier to study emerging SARS-CoV-2 mutants and screen for inhibitors, we developed a simple mini-replicon that produces a minimal background signal, that can be used in any tissue culture lab, and that only requires four small plasmids to work.

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  1. Version published to 10.1101/2021.06.28.450211v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.06.28.450211: (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 nsp7, nsp8 and nsp12 proteins were subsequently detected using the mouse monoclonal anti-DYKDDDDK M2 antibody (Catalogue number F3165, Sigma-Aldrich) diluted 1:1000 in TBST/5% milk.
    anti-DYKDDDDK
    suggested: None
    Cellular proteins were detected using rabbit polyclonal antibodies anti-GAPDH (Catalogue number GTX100118, GeneTex) diluted 1:4000 in TBST/5% milk.
    anti-GAPDH
    suggested: (GeneTex Cat# GTX100118, RRID:AB_1080976)
    Secondary antibodies IRDye 680 goat anti-mouse (Catalogue number 926-68020, Li-cor) and IRDye 800 donkey anti-rabbit (Catalogue number 926-32213, Li-cor) were used to detect western signals with an Odyssey scanner (Li-cor)
    anti-mouse
    suggested: (LI-COR Biosciences Cat# 926-68020, RRID:AB_10706161)
    anti-rabbit
    suggested: (LI-COR Biosciences Cat# 926-32213, RRID:AB_621848)
    Experimental Models: Cell Lines
    SentencesResources
    Cells: Human embryonic kidney (HEK) 293T cells (ATCC CRL-3216) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (PAN-Biotech) with 10% feral calf serum (FCS) (Sigma-Aldrich), 1% L-Glutamine (Sigma-Aldrich).
    HEK
    suggested: None
    Mini genome assay: To measure SARS-CoV-2 polymerase activity in cell culture, approximately 2×105 293T cells were transfected with 200 ng each of pcDNA6.B-nsp7-flag, pcDNA6.B-nsp8-flag, and pcDNA6.B-nsp12-flag, and 25 ng linear pPolI-SARS-CoV2-NLuc-N plasmid using 2µl of Lipofectamine 2000 (Invitrogen) to a DNA:lipofectamine ratio of 1:3 and 100µl Opti-MEM (Gibco).
    293T
    suggested: None
    Luciferase-based IFN expression assay: HEK 293T cells were transfected with SARS-CoV-2 nsp plasmids as described above for the mini-genome assay and co-transfected with 100 ng of a firefly luciferase reporter plasmid under the control of the IFN-β promoter as described previously (48).
    HEK 293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: Plasmids pcDNA6.B-nsp7-flag, pcDNA6.B-nsp8-flag, pcDNA6.B-nsp12-flag, pcDNA6.B-nsp10-flag, pcDNA6.B-nsp14-flag, pcDNA6.B-nsp9-flag, pcDNA6.B-nsp13-flag
    pcDNA6.B-nsp8-flag
    suggested: None
    pcDNA6.B-nsp10-flag
    suggested: None
    pcDNA6.B-nsp14-flag
    suggested: None
    pcDNA6.B-nsp9-flag
    suggested: None
    , pcDNA6.B-nsp16-flag containing codon-optimized versions of SARS-CoV-2 strain Wuhan-Hu-1 (2019) nsp7,
    pcDNA6.B-nsp16-flag
    suggested: None
    , motif C (DD761-762AA), P323L, P322L), in nsp7 (S25L, S26F), and in nsp8 (M129I, D163L) were introduced by site-directed mutagenesis of pcDNA6.B-nsp12-flag, pcDNA6.B-nsp7-flag or pcDNA6.B-nsp8-flag using primers listed in Table 1.
    pcDNA6.B-nsp12-flag
    suggested: None
    To construct the negative-sense RNA reporter, we designed construct PolI-SARS-CoV2-NLuc-N-HDR and ordered this as insert in the pMK-RQ backbone (Invitrogen construct ID 20ACX40P).
    pMK-RQ
    suggested: None
    Transfection control pcDNA3-FF-luciferase and IFN-β reporter plasmid pIFΔ(−116)lucter were described previously (48, 49).
    pcDNA3-FF-luciferase
    suggested: None
    Mini genome assay: To measure SARS-CoV-2 polymerase activity in cell culture, approximately 2×105 293T cells were transfected with 200 ng each of pcDNA6.B-nsp7-flag, pcDNA6.B-nsp8-flag, and pcDNA6.B-nsp12-flag, and 25 ng linear pPolI-SARS-CoV2-NLuc-N plasmid using 2µl of Lipofectamine 2000 (Invitrogen) to a DNA:lipofectamine ratio of 1:3 and 100µl Opti-MEM (Gibco).
    pcDNA6.B-nsp7-flag
    suggested: None
    pPolI-SARS-CoV2-NLuc-N
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis: Statistical analysis was performed with GraphPad PRISM 8 software.
    GraphPad PRISM
    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.06.28.450211v1 on bioRxiv