Glycans located upstream of the furin cleavage site in SARS-CoV-2 Spike protein influence its processing and functions

Abstract

The SARS-CoV-2 Spike (S) glycoprotein, which plays a central role in viral entry, consists of two subunits: S1, which mediates binding to the ACE2 receptor; and S2, which induces fusion of the viral envelope with the cell membrane. The S glycoprotein also contains a polybasic motif at the S1/S2 junction that is recognised by the furin-type protease family, modulating its entry functions. Several of the 22 N-glycans that decorate S are located near the S1/S2 boundary and are likely to influence S cleavage, which is crucial for inducing fusion—a key step in viral entry. This study characterised the importance of the N-glycans N603, N616, and N657 for S cleavage and its ability to induce fusion and entry. Our data revealed that S harbouring mutations at these three sites relies on a basic residue at position 634 for cleavage. Furthermore, while these N-glycans and R634 were necessary for the ability of S to mediate fusion and entry in the G614 and D614 variants, mutations in these residues had a milder impact in the XBB.1.5 Omicron variant. Consequently, the roles of N603, N616, N657 and R634 in S cleavage, fusion induction and viral entry have evolved alongside these different variants.