A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

  1. Pehuén Pereyra Gerber
  2. Lidia M. Duncan
  3. Edward JD Greenwood
  4. Sara Marelli
  5. Adi Naamati
  6. Ana Teixeira-Silva
  7. Thomas WM Crozier
  8. Ildar Gabaev
  9. Jun R. Zhan
  10. Thomas E. Mulroney
  11. Emily C. Horner
  12. Rainer Doffinger
  13. Anne E. Willis
  14. James ED Thaventhiran
  15. Anna V. Protasio
  16. Nicholas J. Matheson

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Abstract

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

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  1. Version published to 10.1371/journal.ppat.1010265
  2. Version published to 10.1101/2021.03.22.435957v3 on bioRxiv
  3. Version published to 10.1101/2021.03.22.435957v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2021.03.22.435957: (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 AuthenticationAuthentication: Key resources table: Cell culture: HEK293T cells (a kind gift from Paul Lehner, authenticated by STR profiling (Menzies et al., 2018; Miles et al., 2017)) and VeroE6 cells (a kind gift from Rupert Beale, authenticated by species-specific PCR (IDEXX BioAnalytics)) were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in 5% CO2.
    Contamination: All cells were regularly screened and confirmed to be mycoplasma negative (Lonza MycoAlert and IDEXX BioAnalytics).

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were then permeabilised with Perm/Wash buffer (BD), stained for SARS-CoV-2 spike protein using a mouse monoclonal antibody (GeneTex, GTX632604) for 30 mins at room temperature, washed twice, stained with an anti-mouse Alexa Fluor 594 (AF594) secondary antibody (Jackson ImmunoResearch, #715-585-150) for 30 mins at room temperature, washed extensively, mounted with 200 µL/well of Fluoroshield Mounting Media (Sigma, F6057), and analysed by automated microscopy.
    anti-mouse
    suggested: (Jackson ImmunoResearch Labs Cat# 715-585-150, RRID:AB_2340854)
    Cells were then permeabilised with Perm/Wash buffer (BD), stained for SARS-CoV-2 spike protein using a mouse monoclonal antibody (GeneTex, GTX632604) for 30 mins at room temperature, washed twice, stained with an anti-mouse AF647 secondary antibody (Jackson ImmunoResearch, #715-605-150) for 30 mins at room temperature, mounted with 200 µL/well of Fluoroshield Mounting Media (Sigma, F6057), and analysed by confocal microscopy using a Zeiss LSM 710 Inverted confocal microscope equipped with 405, 458, 543 and 633 nm lasers and a Plan Apochromat 63X/1.40 Oil DIC M27 objective.
    anti-mouse AF647
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Key resources table: Cell culture: HEK293T cells (a kind gift from Paul Lehner, authenticated by STR profiling (Menzies et al., 2018; Miles et al., 2017)) and VeroE6 cells (a kind gift from Rupert Beale, authenticated by species-specific PCR (IDEXX BioAnalytics)) were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 C in 5% CO2.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Vectors for transgene expression: Generation of HEK293T-ACE2 and HEK293T-ACE2-30F-PLP2 cells: For transduction of HEK293T cells with ACE2, a pHRSIN-ACE2-Hygro lentiviral stock was generated by co-transfection of HEK293T cells with psPAX2 and pCMV-VSV-G using standard methods.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Flow cytometric analysis of spike protein expression in SARS-CoV-2-infected cells: HEK293T-ACE2 cells were seeded at a density of 9 x 104 cells/48-well in 250 µL complete media.
    HEK293T-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    The ratio of FlipGFP/mCherry mean fluorescence intensity (MFI) in BFP+ (transfected) cells was used to quantitate reporter activation (FlowJo 10.7).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    For each reporter construct, the ratio of FlipGFP/mCherry MFI was calculated manually using Fiji (ImageJ), by creating a mask around syncytiated cells that were both spike+ (infected) and mCherry+ (transfected).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Instead, FASTA sequences were downloaded and aligned using MAFFT (Katoh et al., 2002) with default parameters.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Having manually inspected the genomic alignments to identify the regions of interest, we used the ‘extractalign’ function in the European Molecular Biology Open Software Suite (EMBOSS)
    EMBOSS
    suggested: (EMBOSS, RRID:SCR_008493)
    The resulting amino acid sequences were used as input for the WebLogo application (Crooks et al., 2004; Schneider and Stephens, 1990).
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    Statistical analysis: General data manipulation was conducted using Microsoft Excel, and statistical analysis using Graphpad Prism.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    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: We detected the following sentences addressing limitations in the study:
    To overcome these limitations, we therefore generated luciferase-based reporters. We envisioned at least three advantages of this approach: first, luciferase-based assays are typically highly sensitive; second, since the first step in measuring luciferase activity involves lysing cells, the readout is not affected by syncytia formation during viral infection; and third, luciferase-based assays may be readily adapted to high-throughput platforms. Accordingly, compared with FlipGFP, the window for luciferase-based reporter activation was greatly increased in assays of recombinant proteases (139-fold versus 26-fold for Opt3c/MPro reporters, and 74-fold versus 12-fold for PLP2/PLPro reporters) and viral infection (12-fold vs. 3-fold for Opt3c/MPro reporters, and 29-fold versus 3-fold for PLP2/PLPro reporters). Interestingly, whilst our luciferase-based Opt3c/MPro reporter displayed a higher sensitivity for recombinant protease, our PLP2/PLPro reporter performed markedly better during SARS-CoV-2 infection. This may reflect inversion of the expression levels of MPro and PLPro in the different settings, or differential accessibility of the reporters (substrates) to viral proteases in the context of viral infection. Since PLPro of SARS-CoV has deubiquitylation (DUB) activity, and is able to regulate whole cell levels of ubiquitylation during viral infection, we suspect that PLPro of SARS-CoV-2 may also be able to readily access a wide variety of cellular proteins, including cytosolic...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 58, 55 and 47. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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

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  5. Version published to 10.1101/2021.03.22.435957v1 on bioRxiv