Figure 6. ZEBOV trafficking involves early and late endosomes.

(A) ZEBO-VLPs colocalize with vesicles bearing early endosomal antigen-1 (EEA1) shortly after internalization. HEK293T cells were incubated with fluorescently-labeled ZEBO-VLPs (green) for 10 min at 16°C. After washing to remove unbound VLPs, fresh growth medium was added to cells, which were then incubated at 37°C. At the indicated time cells were fixed, permeabilized and stained for EEA1 (red). Nuclei (blue) were stained with DAPI. Images were taken by confocal microscopy using a 100× oil immersion objective lens. A representative image of mid-optical z-section is shown for each time point. (B) Quantitation of VLP colocalization. VLPs colocalized with EEA1 were counted and expressed as percent of total VLPs in image sections. At least 10 images (5–6 cells/image) were analyzed for each sample. Mean ± st.dev. are presented in the data. (C) ZEBOV requires Rab5 and Rab7 function. HEK293T cells were made to express GFP or GFP-tagged forms of DN Rab5, DN Rab7 by plasmid transfection. Twenty-four h post-transfection cells were incubated with wild-type ZEBOV for 48 h. Cells were fixed after 36 h and immunostained for ZEBOV VP40 matrix protein as a marker of infection. Nuclei were stained with DAPI and images were taken by fluorescence microscopy. Image analysis was performed using Cell Profiler software (Broad Inst. MA) as described in methods. The proportion of cells that were expressing each GFP-tagged fusion protein and infected by ZEBOV was calculated as a fraction of the total cell population and averaged for all replicates (>5). Data were normalized to that seen in cells transfected with GFP alone. (D) Rab5 and Rab7 function is necessary for the cell entry step of infection. To determine the step of infection that was affected by each DN protein, entry assays were performed using HEK293Tcells expressing GFP, or GFP-tagged forms of wild-type Rab5, DN Rab5 or DN Rab7. Cells were incubated with VSV-VLP (open bars) or ZEBO-VLP (solid bars) for 3 h. Subsequently, luciferase activity was measured in each sample and expressed relative to that in control (untransfected) cells. The data represents average ± st.dev. of 3 independent experiments, each performed in duplicate.