SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation

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Abstract

Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2′ cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion.

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  1. SciScore for 10.1101/2020.07.25.221135: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: COVID-19 convalescent serum was collected previously (58) in accordance with ethical requirements (ethics committee UK Erlangen, license number AZ. 174_20 B)
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Western blot: Protein expression was analyzed by polyacrylamide gel electrophoresis on 8%-16% precast gradient gels (Thermo) and Western blotting using antibodies to ACE2 (AF933, R&D Systems), c-Myc-epitope (clone 9E10, Santa Cruz Biotechnology), SARS spike (NB100-56578, Novus Biologicals), HIV-1 Gag p24 (clone 749140, R&D), and GAPDH (GenScript) in NETT-G (150 mM NaCl, 5mM EDTA, 50 mM Tris, 0.05% Triton X-100, 0.25% gelatin, pH 7.5) and donkey anti-mouse horseradish peroxidase (HRP)-coupled (Dianova)
    ACE2
    suggested: None
    NB100-56578
    suggested: (Novus Cat# NB100-56578, RRID:AB_838846)
    HIV-1 Gag p24
    suggested: (R and D Systems Cat# MAB7360, RRID:AB_10993570)
    GAPDH
    suggested: (LSBio (LifeSpan Cat# LS-C94067-150, RRID:AB_1932766)
    anti-mouse
    suggested: None
    , goat anti-rabbit HRP-coupled (Life Technologies) or rabbit anti-goat HRP-coupled (Proteintech) secondary antibody in 5% dry milk powder in PBS with 0.05% Tween 20.
    anti-rabbit
    suggested: None
    anti-goat HRP-coupled ( Proteintech )
    suggested: None
    The cells were then incubated in either convalescent serum at 1:1000 dilution or soluble ACE2-Fc fusion protein at 2 ng/μl, both described elsewhere (58), for 1h in 10% FCS in PBS, followed by one wash in a large volume of PBS and then incubation with Alexa647-coupled anti-human secondary antibody (Thermo Fisher Scientific) at 1:200 in 10% FCS in PBS.
    anti-human secondary
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For Calu-3 cells 1mM Sodium-Pyruvate (Thermo Fisher Scientific) was added.
    Calu-3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Flow cytometry: 293T cells were transfected with the respective spike expression constructs.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    SARS-CoV-2 infections: Primary SARS-CoV-2 isolate ER-PR2 was a kind gift from Klaus Überla, Erlangen, and was originally isolated on Vero cells.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Experimental Models: Organisms/Strains
    SentencesResources
    293T effector cells were seeded in a 10 cm dish at 70-80% confluency and transfected with either the Vp16-Gal4 (all experiments except Fig.
    Vp16-Gal4
    suggested: None
    Software and Algorithms
    SentencesResources
    Data was analyzed using Flowing software (version 2.5) and GraphPad Prism, version 6, for Windows (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    These datapoints with the undiluted sample set to 1 was approximated by an exponential function using Microsoft Excel 2020.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04355026RecruitingUse of Bromhexine and Hydroxychloroquine for Treatment of CO…
    NCT04273763Active, not recruitingEvaluating the Efficacy and Safety of Bromhexine Hydrochlori…
    NCT04340349Enrolling by invitationLow-dose Hydroxychloroquine and Bromhexine: a Novel Regimen …


    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.

    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.