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. 2010 Sep 23;6(9):e1001121. doi: 10.1371/journal.ppat.1001121

Figure 2. Internalization of DiI-labeled EBOV particles is independent of the caveolae-mediated endocytic pathway.

Figure 2

(A) DiI-labeled EBOV particles do not co-localize with eGFP-labeled caveolae. DiI-EbolaΔVP30 virions (left panel) or DiI-Ebola VLPs (right panel) were adsorbed to Cav1-eGFP-expressing Vero cells for 30 min on ice. The cells were then incubated for 15 min at 37°C and the co-localization of DiI-labeled viral particles with eGFP-labeled caveolae was analyzed by using confocal laser scanning microscope. Insets show enlargements of the boxed areas. Scale bars, 10 µm. (B) Effect of Cav1 down-regulation on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control siRNA (left panels) or siRNA to down-regulate Cav1 expression (right panels). The efficiency of Cav1 down-regulation was analyzed by use of immunofluorescent staining 48 h post-transfection (lower panels) and western blot analysis (C). 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. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in siRNA-transfected Vero cells. The internalized DiI-virions were analyzed in 10 individual siRNA-transfected cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Internalization of DiI-labeled Ebola virions in cells lacking Cav1. DiI-labeled EbolaΔVP30 virions were adsorbed to Cav1-deficient Huh7 cells for 30 min on ice. The internalization of DiI-EbolaΔVP30 virions was analyzed 2 h after the temperature shift to 37°C. Outlines of individual cells were drawn. Scale bar, 10 µm. (F) Effect of dynasore on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO (left panel) or dynasore (right panel) for 30 min at 37°C. Labeled Ebola VLPs were adsorbed to the cells for 30 min on ice and incubated for 2 h at 37°C in the presence of DMSO or dynasore. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Dynasore treatment interfered with the internalization of Alexa Fluor 633-Tf (green in right panel), attesting to its functionality. Scale bars, 10 µm. (G) Quantitative analysis of the internalization of DiI-labeled Ebola virions in dynasore-treated Vero cells. The internalized DiI-virions were analyzed in 10 individual DMSO- or dynasore-treated cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD.