Beyond ACE2: unveiling the receptor-mediated endocytic dynamic mechanism of SARS-CoV-2 at the single-particle level

Beyond the canonical angiotensin-converting enzyme 2 (ACE2)-mediated entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells, basigin (BSG) and dipeptidyl peptidase 4 (DPP4) may also act as alternative receptors that can facilitate viral attachment and subsequent endocytosis. Herein, we analyzed the binding efficiency and binding dynamics of SARS-CoV-2 virus-like particles (VLPs), mediated by BSG and DPP4 at the single-particle level, by combining super-resolution direct stochastic optical reconstruction microscopy (dSTORM) and single-molecule force spectroscopy (SMFS). dSTORM imaging demonstrated that DPP4 exhibits higher surface expression levels than BSG on HepG2 cells. However, SARS-CoV-2 VLPs display a distinct preference for BSG-dependent cellular entry. Using SMFS, we further probed the binding dynamics between the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) and these two receptors. The results demonstrated that RBD-BSG exhibits greater stability and a more rapid formation rate compared to RBD-DPP4. Subsequently, a real-time force tracing technique was used to show that BSG-mediated VLP entry requires lower force and faster speed compared with DPP4-mediated entry. In particular, we confirmed that the structural integrity of intramolecular disulfide bonds within the RBD is indispensable for mediating high-affinity receptor binding. These findings elucidate the distinct receptor-mediated endocytic dynamic mechanisms underlying SARS-CoV-2 entry mediated by BSG and DPP4, expanding our understanding of viral invasion beyond the ACE2 pathway and providing insights into potential intervention strategies targeting viral infection.