Pharmacological perturbation of intracellular dynamics as a SARS-CoV-2 antiviral strategy

  1. William Bakhache
  2. Emma Partiot
  3. Vincent Lucansky
  4. Yonis Bare
  5. Boris Bonaventure
  6. Caroline Goujon
  7. Cédric Bories
  8. Maika S. Deffieu
  9. Raphael Gaudin

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Abstract

SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.

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  1. ScreenIT

    SciScore for 10.1101/2021.09.10.459410: (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 AuthenticationContamination: Upon sorting, the cells were tested negative for mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and reagents: Mouse anti-Spike and anti-GAPDH were purchased from Genetex and Rabbit anti-N from Sino Biological (Clinisciences).
    anti-Spike
    suggested: None
    anti-GAPDH
    suggested: None
    anti-N
    suggested: None
    The mouse anti-dsRNA antibody (J2) was from Jena Bioscience and rabbit anti-Calnexin from Elabscience.
    anti-dsRNA
    suggested: None
    anti-Calnexin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and primary cells: Huh7.5.1 (Zhong et al., 2005), Vero E6 (ECACC #85020206), Caco2, and HEK 293T cells were cultured in DMEM GlutaMAX (Thermo Fisher Scientific) supplemented with 10% fetal calf serum and penicillin-streptomycin (500 μg/ml; GIBCO).
    Caco2
    suggested: None
    HEK 293T
    suggested: None
    Vials of the Vero E6 N-Ruby3 stable cell line were conserved in liquid nitrogen in 10% DMSO.
    Vero E6
    suggested: None
    N-Ruby3
    suggested: None
    Infections of 50 000 cells were performed at MOI 2 for HCoV-229E-Renilla (as measured on Huh7.5.1 cells) and infection efficiency was analyzed one day later by measuring Renilla activity (Renilla Luciferase Assay System,
    Huh7.5.1
    suggested: RRID:CVCL_E049)
    ZIKV was amplified in Vero cells for 72 h in DMEM GlutaMAX (Gibco) supplemented with 2% fetal bovine serum (FBS; Dominique Dutscher), 10 mM HEPES (Thermo Fisher Scientific) and 1X penicillin-streptomycin (Gibco).
    Vero
    suggested: None
    Recombinant DNA
    SentencesResources
    Bajar et al, Sci Rep, 2016), N was firstly amplified by PCR from the plasmid pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro (addgene#141391) using the sense primer 5’ TTGCGGCCGCGCCACCATGAGCGATAACG-3’ (NotI site underlined) and the antisense primer 5’-CGCCTGAGTAGAATCGGCT-3 (overlapped region underlined).
    pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro
    suggested: RRID:Addgene_141391)
    The resulting PCR product was ligated into the digested NotI and BamHI lentiviral plasmid pHAGE IRES puro.
    pHAGE IRES puro
    suggested: RRID:Addgene_122202)
    The resulting plasmid pHAGE N-mRuby3 IRES puro is available on Addgene (#170466).
    pHAGE N-mRuby3
    suggested: None
    In parallel, a pHAGE N-mNeonGreen IRES puro was also generated (Addgene #170467).
    pHAGE N-mNeonGreen IRES
    suggested: None
    Generation of stable cell line: HEK 293T cells were transfected with pxPAX2, VSV-G and pHAGE N-mRuby3 IRES puro using JetPrime
    pHAGE N-mRuby3 IRES puro
    suggested: None
    Software and Algorithms
    SentencesResources
    Live cell imaging: Image acquisition was performed on an AxioObserver.Z1 inverted microscope (Zeiss) mounted with a CSU-X1 spinning disk head (Yokogawa), a back-illuminated EMCCD camera (Evolve, Photometrics) and a ×100, 1.4 NA oil objective (Zeiss) controlled by MetaMoprh.
    MetaMoprh
    suggested: None
    Images were processed using Fiji (ImageJ software version 1.51h) and quantitative analyses were performed using Bitplane Imaris x64 version 9.2.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    The mean and standard deviation (SD) of the mean were plotted using GraphPad Prism version 7.04.
    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:
    • 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.

  2. Version published to 10.1101/2021.09.10.459410v2 on bioRxiv
  3. Version published to 10.1101/2021.09.10.459410v1 on bioRxiv