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
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Abstract

The spîke (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic “priming” at P<underline>R</underline>RA<underline>R</underline>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<underline>KR</underline>815↓ was strongly enhanced by ACE2, but not for the optimized S2’ K<underline>RR</underline>KR815↓ 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.

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