Proteolytic activation of SARS-CoV-2 spike at the S1/S2 boundary: potential role of proteases beyond furin

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2’ site is known to activate the S protein for viral entry, which can occur at either the cell plasma membrane or the endosomal membrane. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which expands viral tropism to lung cells. Here, we analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the implications of SARS-CoV-2’s unique insert on its origin. We also utilized viral pseudoparticles to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for plasma membrane entry into Calu-3 cells, a model lung epithelial cell line, but not for endosomal entry Vero E6 cells, a model cell culture line, and that other proteases in addition to furin are responsible for processing SARS-CoV-2 S1/S2.

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

“…Previous work has shown that treatment of MERS-infected Calu-3 cells with E64d, a broad cysteine protease inhibitor, had diminished efficacy presumably as a result of low CTSL expression but heightened TMPRSS2 expression. This shift in expression profile resulted in the predominant processing of the MERS spike protein by TMPRSS2 (38). It was also suggested that cleavage of the SARS-CoV-2 spike protein by TMPRSS2 in cells with low CTSL levels (such as Calu-3) is essential for efficient viral entry, whereas in TMPRSS2 negative cells (Vero E6) viral infection can proceed with high efficiency using CTSL (39).…”

Role of Serine Proteases and Host Cell Receptors Involved in Proteolytic Activation, Entry of SARS-CoV-2 and Its Current Therapeutic Options

“… 60 Tang and his coworkers in their preprint studies in 2020 identified the presence of additional amino acid residue, P-R-R-A in SARS-CoV-2 at S1/S2 site that was not notified in SARS-CoV. 61 R-R-A-R amino acid residue at the S1/S2 site of S protein may be sensitive to furin dependent cleavage. 62 The activity of furin resides on this poly basic motif to activate the degradation of S protein to generate S2 and S1 subunits.…”

Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

“…Furin proteolysis at the S1-S2 boundary (681–685) and in S2 (811–815) exposes the RBD to enable hACE2 binding, and the S2 domain to initiate membrane fusion 5 . Recent studies show that these cleavage sites are not necessarily specific for furin-mediated proteolysis and that S protein may be processed by multiple proteases to open the RBD into the active conformation 2 – 4 , 33 . Consistent with these observations, both the furin proteolysis consensus sites and the arginines that are critical for proteolysis are not conserved in the S protein (Fig.…”

“…Another issue relates to the furin cleavage sites. As the viral S protein activation appears to require furin proteolysis 2 – 4 , protease-specific inhibitors are tested as a means to protect from infection 48 . However, our analysis suggests that this may not be an effective strategy, given the high variability of furin cleavage sites.…”

“…Interaction of SARS-CoV-2 with hACE2 occurs via the Spike (S) protein on the viral envelope. Proteases cleave the S protein into S1 and S2 subunits 2 – 4 to enable viral binding to hACE2 5 and viral entry by membrane fusion 6 . The S protein is a homotrimer and the S1 subunit of each of the monomers of the S protein contains the receptor-binding domain (RBD; Fig.…”