Molecular Analysis of SARS-CoV-2 Spike Protein-Induced Endothelial Cell Permeability and vWF Secretion.

Coronavirus disease COVID-19, which is caused by severe acute respiratory syndrome coronavirus SARS-CoV-2, has become a worldwide pandemic in recent years. In addition to being a respiratory disease, COVID-19 is a 'vascular disease' since it causes a leaky vascular barrier and increases blood clotting by elevating von Willebrand factor (vWF) levels in the blood. In this study, we analyzed in vitro how the SARS-CoV-2 spike protein S1 induces endothelial cell (EC) permeability and its vWF secretion, and the underlying molecular mechanism for it. We showed that the SARS-CoV-2 spike protein S1 receptor-binding domain (RBD) is sufficient to induce endothelial permeability and vWF-secretion through the angiotensin-converting enzyme (ACE)2 in an ADP-ribosylation factor (ARF)6 activation-dependent manner. However, the mutants, including those in South African and South Californian variants of SARS-CoV-2, in the spike protein did not affect its induced EC permeability and vWF secretion. In addition, we have identified a signaling cascade downstream of ACE2 for the SARS-CoV-2 spike protein-induced EC permeability and its vWF secretion by using pharmacological inhibitors. The knowledge gained from this study could be useful in developing novel drugs or repurposing existing drugs for treating infections of SARS-CoV-2, particularly those strains that respond poorly to the existing vaccines.

SARS-CoV-2 pseudovirus enters the host cells through spike protein-CD147 in an Arf6-dependent manner.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants is threatening public health around the world. Endocytosis functions as an important way for viral infection, and SARS-CoV-2 bears no exception. However, the specific endocytic mechanism of SARS-CoV-2 remains unknown. In this study, we used endocytic inhibitors to evaluate the role of different endocytic routes in SARS-CoV-2 pseudovirus infection and found that the viral infection was associated with caveolar/lipid raft- and cytoskeleton-mediated endocytosis, but independent of the clathrin-mediated endocytosis and macropinocytosis. Meanwhile, the knockdown of CD147 and Rab5a in Vero E6 and Huh-7 cells inhibited SARS-CoV-2 pseudovirus infection, and the co-localization of spike protein, CD147, and Rab5a was observed in pseudovirus-infected Vero E6 cells, which was weakened by CD147 silencing, illustrating that SARS-CoV-2 pseudovirus entered the host cells via CD147-mediated endocytosis. Additionally, Arf6 silencing markedly inhibited pseudovirus infection in Vero E6 and Huh-7 cells, while little change was observed in CD147 knockout-Vero E6 cells. This finding indicated Arf6-mediated CD147 trafficking plays a vital role in SARS-CoV-2 entry. Taken together, our findings provide new insights into the CD147-Arf6 axis in mediating SARS-CoV-2 pseudovirus entry into the host cells, and further suggest that blockade of this pathway seems to be a feasible approach to prevent the SARS-CoV-2 infection clinically.