SARS-CoV-2 spike-glycoprotein processing at S1/S2 and S2’and shedding of the ACE2 viral receptor: roles of Furin and TMPRSS2 and implications for viral infectivity and cell-to-cell fusion

  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. Etienne Decroly
  16. Tram NQ Pham
  17. Éric A. Cohen
  18. Nabil G. Seidah

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

The spîke (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic “priming” at P R RA R 685 ↓ into S1 and S2 (cleavage at S1/S2), and “fusion-activation” at a S2’ site for viral entry. In vitro , Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2’, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2’ processing by Furin at KPS KR 815 ↓ was strongly enhanced by ACE2, but not for the optimized S2’ K RR KR 815 ↓ mutant (μS2’), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a “pH-dependent” endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a “pH-independent” viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin (BOS) and TMPRSS2 (Camostat). Consistently, these inhibitors potently reduce infectious viral titer and cytopathic effects, an outcome enhanced when both compounds were combined. Quantitative analyses of cell-to-cell fusion and spîke processing revealed the key importance of the Furin sites for syncytia formation. Our assays showed that TMPRSS2 enhances fusion and proteolysis at S2’ in the absence of cleavage at S1/S2, an effect that is linked to ACE2 shedding by TMPRSS2. Overall, our results indicate that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and in promoting viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2.

IMPORTANCE

SARS-CoV-2 is the etiological agent of COVID-19 that resulted in >5 million deaths. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein is cleaved at two sites: S1/S2 and S2’. Cleavage at S1/S2, induces a conformational change favoring the recognition of ACE2. The S2’ cleavage is critical for cell-to-cell fusion and virus entry into host cells. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a non-toxic Furin inhibitor with a TMPRSS2 inhibitor could significantly reduce viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

Article activity feed

  1. Version published to 10.1101/2020.12.18.423106v3 on bioRxiv
  2. Version published to 10.1101/2020.12.18.423106v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.12.18.423106: (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

    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), or SARS-CoV-2 spike antibody (rabbit polyclonal GenTex GTX135356; 1:2,000; GenTex).
    SARS-CoV-2
    suggested: (GeneTex Cat# GTX135356, RRID:AB_2887482)
    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)
    ACE2
    suggested: (LSBio (LifeSpan Cat# LS-C347-500, RRID:AB_1271966)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and transfection: Monolayers of HeLa, HEK293T, HEK293T17
    HEK293T
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    HEK293T-ACE251, 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-ACE251
    suggested: None
    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.
    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: None
    HeLa cells were transiently transfected with different constructs of SARS-CoV-2 Spike or NL4.3-HIV Env, or an empty vector and 0.2 μg of CMV-Tat
    HeLa
    suggested: None
    HeLa TZM-bl cells were transfected with human ACE2, TMPRSS2 or a combination of both.
    HeLa TZM-bl
    suggested: NIH-ARP Cat# 8129-442, RRID:CVCL_B478)
    Different combinations of HeLa and HeLa-TZM-bl cells were placed in co-culture plate at a ratio of 1:1 for a total of 60,000 cells/well of a 96 well place.
    HeLa-TZM-bl
    suggested: None
    Upon removal of spent media, 293T-ACE2 and Calu-3 cells were gently washed twice with PBS and analyzed for firefly- or nano-luciferase activity, respectively using Promega luciferase assay (Cat # E1501) or Nano-Glo luciferase system (Cat # N1110), respectively.
    Calu-3
    suggested: None
    Plaque assay in Vero E6: Vero E6 cells (1.2 × 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
    Software and Algorithms
    SentencesResources
    The data from two independent experiments done in triplicates was used to calculate the CC50 by nonlinear regression using GraphPad Prism V5.0 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

  4. Version published to 10.1101/2020.12.18.423106v1 on bioRxiv