A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2

  1. Felix Pahmeier
  2. Christopher J. Neufeldt
  3. Berati Cerikan
  4. Vibhu Prasad
  5. Costantin Pape
  6. Vibor Laketa
  7. Alessia Ruggieri
  8. Ralf Bartenschlager
  9. Mirko Cortese

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Abstract

Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence.

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  1. Version published to 10.1128/jvi.01715-20
  2. ScreenIT

    SciScore for 10.1101/2020.08.31.276683: (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

    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and virus strains: HEK-293T, A549 and VeroE6 cells were purchased from ATCC; Huh7 cells (24) were obtained from Heinz Schaller (Center for Molecular Biology,
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Generation of the cell lines Huh7-Lunet and the derivative Huh7-Lunet-T7, stably expressing the RNA polymerase of bacteriophage T7, have been previously described (25, 26).
    Huh7-Lunet
    suggested: RRID:CVCL_U459)
    Except for DENV-faR that was generated by electroporation of BHK-21 cells as previously described (28), all virus stocks were generated by infection of VeroE6 cells.
    BHK-21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    Subconfluent HEK-293T cells were transfected with the pWPI vector encoding the reporter construct together with packaging plasmids pCMV-Gag-Pol and pMD2-VSV-G (kind gifts from D. Trono, EPFL, Lausanne).
    HEK-293T
    suggested: None
    Lentiviral transduction was performed by addition of the filtered supernatant to Huh7, Huh7-Lunet-T7 or A549-ACE2 cells (multiplicity of infection (MOI) = 5) in presence of 4 μg/mL of polybrene.
    A549-ACE2
    suggested: None
    Live cell imaging: Huh7-Lunet-T7 cells expressing the dengue reporter constructs (Lunet-T7-RC) were seeded onto a glass bottom 35 cm2 dish (Mattek) at a density of 2 × 104.
    Huh7-Lunet-T7
    suggested: None
    Software and Algorithms
    SentencesResources
    Cell lines and virus strains: HEK-293T, A549 and VeroE6 cells were purchased from ATCC; Huh7 cells (24) were obtained from Heinz Schaller (Center for Molecular Biology,
    ATCC
    suggested: (ATCC, RRID:SCR_001672)
    Bioinformatics analysis: Images were analyzed using the Fiji software (32, 33)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Graph generation and statistical analysis was performed using the GraphPad Prism 8.1 software package.
    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 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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 10.1101/2020.08.31.276683 on bioRxiv