Figure 6. Macropinocytosis-associated events occur during Ebola virion internalization.

(A) The effect of the internalization of DiI-labeled Ebola VLPs on dextran uptake. Vero cells were incubated with 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K in the absence or presence of Ebola VLPs for 60 min at 37°C. The uptake of Alexa Fluor 647-Dex Mw 10K was analyzed by using flow cytometry. The effect of EIPA pretreatment was assessed in parallel. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (B) Co-localization of internalized DiI-labeled Ebola VLPs and Dex Mw 10K. DiI-Ebola VLPs were adsorbed to Vero cells for 30 min on ice. The cells were cultured in the presence of 0.5 mg/ml Alexa Fluor 647-Dex Mw 10K for 10 min at 37°C. Co-localization of DiI-virions (red) and Alexa Fluor-Dex Mw 10K (green) was analyzed by using confocal laser scanning microscope. Co-localized virions are shown by arrows. Outlines of individual cells were drawn. Scale bar, 10 µm. (C) Effect of a dominant-negative form of Rac1 on the internalization of DiI-labeled Ebola virions. The eGFP-fused, wild-type Rac1 (wtRac1, upper panels) or the dominant-negative form of Rac1 (dnRac1, lower panels) was expressed in Vero cells. DiI-labeled Ebola VLPs were adsorbed to the cells for 30 min on ice. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. Expression of dnRac1 interfered with the internalization of Alexa Fluor 647-Dex Mw 10K (blue; lower middle panel), attesting to its functionality. Scale bars, 10 µm. (D) Quantitative analysis of the internalization of DiI-labeled Ebola virions in wtRac1 or dnRac1-expressed Vero cells. The internalized DiI-virions were measured in 10 individual wtRac1 or dnRac1-expressed cells. Each experiment was performed in triplicate and the results are presented as the mean ± SD. (E) Effect of PKC inhibitors on the internalization of DiI-labeled Ebola virions. Vero cells were treated with DMSO or staurosporine (Stauro) 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 absence or presence of inhibitor. Surface-bound virions were removed by trypsin and the internalization of DiI-virions was analyzed by using confocal laser scanning microscope. The internalized DiI-virions were analyzed in 10 individual DMSO- or staurosporine-treated cells (red bars). The efficiency of Alexa Fluor-Dex Mw 10K uptake in inhibitor-treated cells was measured by using flow cytometry (blue bars). Each experiment was performed in triplicate and relative uptake efficiencies are presented as the mean ± SD (red bars). Staurosporine treatment interfered with the internalization of Alexa Fluor 633-Tf (blue bars), attesting to its functionality. (F) The down-regulation of Cdc42 and Pak1 by siRNA. The efficiencies of Cdc42 and Pak1 knock-down were assessed by use of RT-PCR. Total cellular RNA was isolated from siRNA-transfected Vero cells 48 h post-transfection by using the TRI reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was performed with Molony murine leukemia virus RTase using a random hexamer (Invitrogen) according to the manufacturer's protocol. PCR was carried out for 25–30 cycles consisting of a DNA denaturing step for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C by use of Taq DNA polymerase (Promega). Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an endogenous control. The oligonucleotides used for amplification of individual genes are shown in Table S1. (G) Effect of down-regulation of Cdc42 and Pak1 on the internalization of DiI-labeled Ebola virions. Vero cells were transfected with control (Cont) non-targeting siRNA or siRNA to down-regulate Cdc42 and Pak1 expression. 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 trypsin for 5 min at 37°C and the internalization of Ebola VLPs was analyzed by using confocal laser scanning microscope, and the number of DiI-virions in 10 individual siRNA-transfected cells was measured. The efficiency of Alexa Fluor-Dex Mw 10K uptake in siRNA-transfected cells was measured by use of flow cytometry (blue bars). Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. (H) The internalization of Ebola virions is associated with plasma membrane ruffling. DiI-EbolaΔVP30 virions were adsorbed to eGFP-actin-expressing Vero cells for 30 min on ice. The cells were then incubated at 37°C and time-lapse images were acquired at 15-second intervals over a period of 10 min by using confocal laser scanning microscope. Still frames at the indicated times (sec) after the temperature shift to 37°C are shown. Scale bar, 10 µm.