Fig 3. Trypsin treatment can allow for ACE2-independent cell entry.

A) S protein driven cell entry in the presence and absence of trypsin. Particles bearing the indicated S proteins (or no S protein) were preincubated with or without trypsin (50 μg/ml for 30 min at 37°C) before being added to the respective cell lines. S-protein driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in the cell lysate at 16-18h post inoculation. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates) in which cell entry was normalized against that measured for particles bearing no S protein (set as 1). Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). B) Trypsin treatment of viral particles but not target cells promotes entry. Vero cells or pseudotyped particles bearing the indicated S proteins were pre-incubated with trypsin (50 μg/ml for 30 min at 37°C) and subsequently trypsin inhibitor (200 μg/ml for 10 min at 37°C) as indicated. The pseudotyped particles were added to the cells. S-protein-driven cell entry was analyzed and data presented as described for panel A. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). C) Multiple bat sarbecovirus spike proteins can employ an ACE2-independent entry pathway following exposure to trypsin. Particles bearing the indicated S proteins were incubated with trypsin (50 μg/ml for 30 min at 37°C) before addition onto 293T wildtype (293T WT) and 293T ACE2 knockout cells (293T KO-ACE2). S-protein-driven cell entry was analyzed and data presented as described for panel A. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). Of note, entry of particles bearing VSV-G in the presence of trypsin has not been analyzed (n.t. = not tested).