FIG 2.

Quantitative analysis of protein VI release, membrane lysis, and lysosome association during virus entry. (A) (Top) Quantification of protein VI release over time. Cells were infected with Alexa 488-labeled wt virus (black circles), PVI-L40Q virus (open circles), or PRO-P137L virus (black triangles), and protein VI-positive viruses were identified by antibody staining at the indicated time points. The error bars indicate cell-to-cell variations (n ≥ 100 particles). (Middle) Quantification of galectin 3 association of viruses over time. Cells were infected with Alexa 488-labeled viruses as for the top graph, fixed, and stained for endogenous galectin 3 at the indicated time points. The error bars indicate cell-to-cell variations (n ≥ 100 particles). (Bottom) Quantification of virus association with lamp-2. Cells were infected with Alexa 488-labeled virus as for the top graph, and association of virus with lamp-2 was quantified at each time point. The error bars indicate cell-to-cell variations (n ≥ 100 particles). (B) Immunofluorescence analysis of virus association with lamp-2. Cells were infected with Alexa 488-labeled wt virus (top), PRO-P137L virus (middle), and PVI-L40Q virus (bottom) for 1 h and stained for lamp-2 association. Viruses are depicted in green, lamp-2 in red, and the DAPI signal in gray. Enlargements of the dashed boxes are depicted on the right. Clusters of virus colocalizing with lamp-2 appear yellow and are indicated by arrows. Scale bars, 10 μm.