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

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic "priming" at PRRAR685{downarrow} into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at KPSKR815{downarrow} (cleavage at S2) for viral entry. Both cleavages occur at Furin-like motifs suggesting that proprotein convertases might promote virus entry. In vitro Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than S2, whereas TMPRSS2 cleaved at both sites. In HeLa cells endogenous Furin-like enzymes cleave mainly at S1/S2 during intracellular protein trafficking, as confirmed by mutagenesis. We also mapped the S2 cleavage site by proteomics and further showed that S2-processing by Furin, while limited, was strongly enhanced in the presence of ACE2. In contrast, the S2 KRRKR815{downarrow} mutant (S2) was considerably better cleaved by Furin, whereas individual/double KR815AA mutants are retained in the endoplasmic reticulum (ER). Pharmacological inhibitors of convertases (Boston Pharmaceuticals - BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. However, under co-expression the S-protein was prematurely cleaved by TMPRSS2 into ER-retained, non-O-glycosylated S2 and S2 products. Quantitative analysis of cell-to-cell fusion and Spike processing using Hela cells revealed the key importance of the Furin sites for syncytia formation and unveiled the enhanced fusogenic potential of the - and {delta}-variants of the S-protein of SARS-CoV-2. Our fusion assay indicated that TMPRSS2 enhances S2 formation, especially in the absence of Furin cleavage, as well as ACE2 shedding. Furthermore, we provide evidence using pseudoparticles 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 and TMPRSS2. Consistently, in Calu-3 cells BOS- inhibitors or Camostat potently reduce infectious viral titer and cytopathic effects and this outcome was enhanced when both compounds were combined. Overall, our results show that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and promote viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. Author SummarySARS-CoV-2 is the etiological agent of the COVID-19 pandemic that spread around the world in 2020 resulting in [~]4 million deaths. The surface 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 needs to be cleaved at two sites: S1/S2 and S2. Cleavage at S1/S2 induces a conformational change in the spike protein favouring the recognition of the ACE2 receptor. The S2 cleavage is critical for the exposure of the fusion domain of the S-protein needed for virus entry into host cells. Several new S-variants contain mutations in the S1/S2 cleavage site possibly enhancing the S-protein processing, especially for the {delta}-variant, which may explain the higher transmissibility of this virus worldwide. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 in SARS-CoV-2 entry and suggest that the combination of a non-toxic Furin inhibitor with that of TMPRSS2 could significantly reduce viral entry in lung cells, as evidenced by our results showing 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.