Figure 7. Macropinocytotic internalization of Ebola virions is GP-dependent.

(A) Co-localization of SNX5 with VSV pseudotyped with EBOV GP. Labeled VSV particles pseudotyped with EBOV GP (DiI-VSVΔ*G-GP) or VSV G (DiI-VSVΔ*G-G) were adsorbed to eGFP-SNX5-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 20-second intervals over a period of 30 min by using confocal laser scanning microscope. Still frames of DiI-VSVΔ*G-GP (left panel) and DiI-VSVΔ*G-G (right panel) at 10 min after the temperature shift are shown. DiI-pseudovirions that co-localize with eGFP-SNX5 are indicated by arrows. Scale bars, 10 µm. (B) Graphic representation of the co-localization of EBOV GP-pseudotyped VSV virions with Rab7-positive vesicles. Co-localization of DiI-VSVΔ*G-GP (green bars) with Rab7-positive vesicles was analyzed at the indicated time points as indicated in the Materials and Methods. Experiments were performed in triplicate and the results are presented as the mean ± standard deviation. Results obtained for DiI-EbolaΔVP30 (blue bars) and DiI-VSVΔ*G-G (red bars) are shown for comparison. (C) Effect of macropinocytosis inhibitors on the co-localization of DiI-labeled VSV pseudovirions with eGFP-Rab7-positive vesicles. Vero cells expressing eGFP-Rab7 were pretreated with CytoD, Wort, LY294002 or EIPA for 30 min at 37°C; control cells were treated with DMSO. DiI-labeled VSVΔ*G-GP (green bars) or VSVΔ*G-G (red bars) were adsorbed to cells for 30 min on ice. The cells were then incubated at 37°C in the presence of inhibitors for 2 h. Co-localization of DiI-pseudovirions with eGFP-Rab7-positive vesicles was analyzed as described in the Materials and Methods. Experiments were carried out in triplicate and the results are presented as the mean ± standard deviation. (D) Effect of macropinocytosis inhibitors on the infectivity of VSV pseudovirions. Vero cells were treated with individual inhibitors for 30 min at 37°C and infected with VSVΔG*-GP (green bars) or VSVΔG*-G (red bars) in the presence of the inhibitor. 1 h post-infection, surface-bound virions were removed by trypsin and the cells were cultured for 24 h in the absence of inhibitors. The infection efficiency of each pseudovirus was determined by measuring the number of GFP-positive cells using with conventional fluorescent microscope. Each experiment was performed in triplicate and the relative infection efficiencies are presented as the mean ± SD.