Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2’ by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2

  1. Rachid Essalmani
  2. Jaspreet Jain
  3. Delia Susan-Resiga
  4. Ursula Andréo
  5. Alexandra Evagelidis
  6. Rabeb Mouna Derbali
  7. David N. Huynh
  8. Frédéric Dallaire
  9. Mélanie Laporte
  10. Adrien Delpal
  11. Priscila Sutto-Ortiz
  12. Bruno Coutard
  13. Claudine Mapa
  14. Keith Wilcoxen
  15. Étienne Decroly
  16. Tram NQ Pham
  17. Éric A. Cohen
  18. Nabil G. Seidah

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Abstract

The author has withdrawn this manuscript due to a duplicate posting of manuscript number 423106. Therefore, the author does not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author (Nabil G. Seidah at seidahn@ircm.qc.ca .

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  1. Version published to 10.1101/2021.07.02.450896v2 on bioRxiv
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    SciScore for 10.1101/2021.07.02.450896: (What is this?)

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

    Table 1: Rigor

    EthicsEuthanasia Agents: Inhibitor treatment: At 24h post transfection, cells were incubated for 6h with two pan-PC inhibitors: the cell permeable decanoyl-RVKR-chloromethylketone (cmk; 50 mM; 4026850.001; Bachem), or with the cell surface PC-inhibitor hexa-D-arginine (D6R; 20 μM; 344931; EMD).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The proteins were revealed using a V5-monoclonal antibody (V5-mAb V2660; 1:5000; Invitrogen), ACE2 antibody (rabbit monoclonal ab108252; 1:3,000; Abcam),
    V5-mAb
    suggested: None
    V2660
    suggested: None
    TMPRSS2 antibody (rabbit polyclonal; 14427-1-AP; 1:1,000; Proteintech)
    TMPRSS2
    suggested: None
    Actin antibody (rabbit polyclonal A2066; 1:5,000; Sigma), HA-HRP antibody (12-013-819; 1:3,500; Roche), or SARS-CoV-2 spike antibody (rabbit polyclonal GenTex GTX135356; 1:2,000; GenTex).
    HA-HRP
    suggested: None
    SARS-CoV-2
    suggested: (GeneTex Cat# GTX135356, RRID:AB_2887482)
    Namely, 0.3 ml of media were immunoprecipitated with either 25 µl EZ view Red anti-HA affinity gel (E 6779; Sigma-Aldrich) or 1.2 µg ACE2 antibody (goat ployclonal AF 933; R&D) and 30 µl TrueBlot anti-Goat Ig IP beads (00-8844-25; Rockland Inc.), respectively, according to the manufacturers’ protocol.
    anti-HA
    suggested: (Sigma-Aldrich Cat# E6779, RRID:AB_10109562)
    ACE2
    suggested: None
    anti-Goat
    suggested: (Rockland Cat# 00-8844-25, RRID:AB_2610705)
    Upon SDS-PAGE separation and PVDF transfer, the immunoprecipitated proteins were detected using an HA-HRP antibody (12-013-819; 1:3,500; Roche) or ACE2 antibody (rabbit monoclonal ab108252; 1:3,000; Abcam) and TrueBlot anti-Goat IgG HRP (18-8814-31; Rockland Inc.), respectively. Microscopy: To establish the luciferase assay, cell co-cultures were plated on glass coverslips.
    anti-Goat IgG
    suggested: (Rockland Cat# 18-8814-31, RRID:AB_2610843)
    Cells were incubated with primary antibodies overnight at 4°C using an antibody against V5 (mouse monoclonal R960-25; 1:1000; Invitrogen), Spike (mouse monoclonal GTX632604; 1:500; GeneTex) and ACE2 (goat polyclonal AF933; 1:500; RnDsystems).
    V5
    suggested: (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564)
    Experimental Models: Cell Lines
    SentencesResources
    Enzymatic PC-inhibition by BOS-inhibitors: Biochemical assay: The proprotein convertases Furin (108-574-Tev-Flag-6His), PC5A (PCSK5; 115-63-Tev-Flag-6His), PACE4 (PCSK6; 150-693-Tev-Flag-6His), and PC7 (PCSK7; 142-634-Tev-Flag-6His) enzymes were purified from BacMam transduced CHO cells.
    CHO
    suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
    Cellular assay: Analyses were initiated by the addition of U2OS cells simultaneously transduced with a BacMam-delivered construct containing a Golgi-targeting sequence followed by a 12-amino acid Furin/PCSK cleavage site from Bone Morphogenic Protein 10 (BMP10) and then GFP at the C terminus.
    U2OS
    suggested: None
    Cell culture and transfection: Monolayers of HeLa, HEK293T, HEK293T17
    HEK293T
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    , Vero E6 and Calu-3 cells were cultured in 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Wisent) supplemented with 10% (v/v)
    Calu-3
    suggested: None
    HEK293T-ACE2[98], a generous gift from Dr. Paul Bieniasz, were maintained in DMEM containing 10% FBS, 1% nonessential amino acids (NEAA) and 50 µg/ml blasticidin (Invivogen)
    HEK293T-ACE2
    suggested: None
    CHO-ldlD cells, which are defective in UDP-Gal/UDP-GalNAc 4-epimerase [50] and therefore cannot O-glycosylate proteins, and their parental CHO-K1 cells were cultured in DMEM/F12 medium (50/50) (Wisent) supplemented with 3% FBS and F12K medium (Wisent) containing 10% FBS, respectively.
    CHO-K1
    suggested: None
    In certain experiments, 293T17 cells were treated with BOS inhibitors at 6 h post transfection.
    293T17
    suggested: None
    Cell viability assay using MTT: Cells, seeded in a 96-well plate, the day before, at 10,000 (HEK-293T and Vero E6) or 50,000 (Calu-3) cells, were treated with serial 10-fold dilutions of BOS inhibitors for up to 48h.
    HEK-293T
    suggested: RRID:CVCL_A7UK)
    Cell-to-cell fusion assay: HeLa or HeLa TZM-bl cells were plated at 200,000 cells in 12-well plates.
    HeLa TZM-bl
    suggested: NIH-ARP Cat# 8129-442, RRID:CVCL_B478)
    The relative light units (RLU) were measured using a Promega GLOMAX plate reader (Promega, Madison, WI, USA) and values were reported as fold increase over the RLU measured in co-culture of HeLa cells transfected EV with respective TZM-bl cells.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Protein immunoprecipitation from co-culture media: When indicated, the secreted form of double tagged Spike glycoprotein of Sars-CoV-2 (N-terminal HA tag and C-terminal V5 tag) and the TMPRSS2-shed forms of ACE2 receptor from the FBS containing media of co-cultured HeLA and HeLa-TZM-bl cells were immunoprecipitated for WB analyses.
    HeLa-TZM-bl
    suggested: None
    Plaque assay in Vero E6: Vero E6 cells (1.2 x 105 cells/well) were seeded in quadruplicate in 24-well tissue culture plates in DMEM supplemented with 10% FBS two days before infection.
    Vero E6
    suggested: None
    Viral production in the supernatant was quantified using a plaque assay on Vero E6.1 cells as described above.
    Vero E6.1
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: Single tagged (C-terminal V5 tag) or double tagged (N-terminal HA tag and C-terminal V5 tag) Spike glycoprotein of SARS-CoV-2 (optimized sequence) and its mutants were cloned into the pIRES2-EGFP vector.
    pIRES2-EGFP
    suggested: RRID:Addgene_14998)
    The plasmids pCI-NEO-hACE2 received from DW Lambert (University of Leeds) and pIRES-NEO3-hTMPRSS2 from P Jolicoeur (IRCM).
    pCI-NEO-hACE2
    suggested: None
    Luc.R-E-) was obtained from Dr. Nathaniel Landau through the NIH AIDS Reagent Program whereas the pHIV-1NL4-3 ΔEnv-NanoLuc construct was a kind gift from Dr. P
    pHIV-1NL4-3 ΔEnv-NanoLuc
    suggested: None
    Plasmids encoding VSV-G, as HIV-1 Env and tat were previously described [96, 97].
    VSV-G
    suggested: RRID:Addgene_138479)
    To generate HIV particles pseudotyped with SARS-CoV-2 S, 293T17 cells (600,000 cells plated in a 6-well vessel) were transfected with 1 µg pNL4-3.
    pNL4-3
    suggested: None
    Luc.R-E- (or pHIV-1NLΔEnv-NanoLuc) in the presence or absence of 0.3 µg pIR-2019-nCoV-S V5 plasmids using Lipofectamine-3000 (Life Technologies).
    pHIV-1NLΔEnv-NanoLuc
    suggested: None
    V5
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were visualized using a confocal laser-scanning microscope (LSM710, Carl Zeiss) with Plan-Apochromat 63x/1.40 Oil DIC M27 objective on ZEN software.
    ZEN
    suggested: None
    Plaques were counted manually and in parallel, imaged plaque plates were processed and plaques enumerated using an automated algorithm based Matlab software.
    Matlab
    suggested: (MATLAB, RRID:SCR_001622)
    The results from experiments done with two biological replicates and two technical replicates in triplicates were used to calculate the IC50 by nonlinear regression using GraphPad Prism V5.0 software.
    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

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  3. Version published to 10.1101/2021.07.02.450896v1 on bioRxiv