Figure 1. Internalization of DiI-labeled Ebola virions is independent of the clathrin -mediated endocytic pathway.

(A) DiI-labeled Ebola virions (red) do not co-localize with eGFP-labeled CCPs. DiI-EbolaΔVP30 virions (left panel) or DiI-Ebola VLPs (right panel) were adsorbed to Vero cells expressing CLCa-eGFP for 30 min on ice. Cells were then incubated for 15 min at 37°C and the co-localization of DiI-labeled viral particles with eGFP-labeled CCPs was analyzed by using confocal microscope. Insets show enlargements of the boxed areas. Scale bars, 10 µm. (B) Effect of clathrin-heavy chain down-regulation on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control siRNA (left panels) or CHC siRNA (right panels) to down-regulate CHC expression. The efficiency of CHC down-regulation was analyzed by immunofluorescent staining 48 h post-transfection (red; lower panels); the effect of siRNA on Alexa Fluor 633-Tf is apparent (green; lower panels). Labeled Ebola VLPs were adsorbed to the siRNA-transfected cells for 30 min on ice 48 h post-transfection. After incubation for 2 h at 37°C, surface-bound virions were removed by the addition of trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope (upper panels). Outlines of individual cells were drawn. Scale bars, 10 µm. (C) Quantitative analysis of the internalization of DiI-labeled Ebola virions in siRNA-transfected Vero cells. The number of DiI-virions in 10 individual siRNA-transfected cells was measured. Each experiment was performed in triplicate and the results are presented as the mean ± SD.