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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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 Statement IRB: COVID-19 convalescent serum was collected previously (58) in accordance with ethical requirements (ethics committee UK Erlangen, license number AZ. 174_20 B) Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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 … 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 Statement IRB: COVID-19 convalescent serum was collected previously (58) in accordance with ethical requirements (ethics committee UK Erlangen, license number AZ. 174_20 B) Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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) ACE2suggested: NoneNB100-56578suggested: (Novus Cat# NB100-56578, RRID:AB_838846)HIV-1 Gag p24suggested: (R and D Systems Cat# MAB7360, RRID:AB_10993570)GAPDHsuggested: (LSBio (LifeSpan Cat# LS-C94067-150, RRID:AB_1932766)anti-mousesuggested: 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-rabbitsuggested: Noneanti-goat HRP-coupled ( Proteintech )suggested: NoneThe 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 secondarysuggested: NoneExperimental Models: Cell Lines Sentences Resources For Calu-3 cells 1mM Sodium-Pyruvate (Thermo Fisher Scientific) was added. Calu-3suggested: KCLB Cat# 30055, RRID:CVCL_0609)Flow cytometry: 293T cells were transfected with the respective spike expression constructs. 293Tsuggested: 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. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Experimental Models: Organisms/Strains Sentences Resources 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-Gal4suggested: NoneSoftware and Algorithms Sentences Resources Data was analyzed using Flowing software (version 2.5) and GraphPad Prism, version 6, for Windows (GraphPad Software). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)GraphPadsuggested: (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 Excelsuggested: (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:
Identifier Status Title NCT04355026 Recruiting Use of Bromhexine and Hydroxychloroquine for Treatment of CO… NCT04273763 Active, not recruiting Evaluating the Efficacy and Safety of Bromhexine Hydrochlori… NCT04340349 Enrolling by invitation Low-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.
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