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

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic ″priming″ at PRRAR685↓ into S1 and S2 (cleavage at S1/S2), and ″fusion-activation″ at KPSKR815↓ (cleavage at S2′) 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 KPSKR815↓ was strongly enhanced by ACE2, but not for the optimized S2′ KRRKR815↓ 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 spike 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.