Figure 1. Time-course of α2β1-integrin internalization and viral uncoating upon EV1 infection.

(A) Time-lapse confocal imaging of α2β1-integrin uptake for 15 minutes (upper panels), 30 minutes (middle panels) and 2 hours (lower panels) p.i. The uptake was stimulated by using antibodies (a, b, c) or EV1 (d, e, f) to cluster α2β1-integrin in SAOS-α2β1 cells. As a control for integrin clustering, α2β1-integrin distribution was followed for the same time periods after binding of non-clustering monovalent fluorescent Fab fragments (g, h, i), Scale bars: 20 µm. (B) Time-lapse uncoating status of EV1 evaluated by light-sensitive neutral red labeled virus. SAOS-α2β1 cells were treated with bright light for 10 minutes at indicated time points p.i. that rendered viral genome inside the capsid uninfectious and prevented further uncoating. At 7 hours p.i., the cells were fixed with 4% paraformaldehyde, stained with EV1 antiserum and the proportion of infected cells of the total cell number was calculated. Control cells were kept in darkness during the whole infection time interval. Example images are given for cells kept in the darkness for the whole period of 7 h (“DARK”) and for the cells given a 10 min light pulse (“LIGHT”) 10 min p.i. leading to a total block in infection.