Figure 4. ZEBOV uptake and infection is inhibited by EIPA and VLP uptake is associated with dextran containing vesicles.

(A) EIPA inhibits dextran accumulation into vesicles. Vero cells were treated with DMSO or EIPA (50 uM) for 30 min. Subsequently, cells were incubated with Alexafluor₅₉₄-labeled dextran (1 mg/ml) in the presence of the inhibitor. After 30 min, cells were washed, fixed and observed by confocal microscopy. Nuclei (blue) were stained with DAPI. Images were taken using a 100× oil immersion objective lens. (B) Accumulation of dextran in cells was analyzed by counting the total number of macropinocytic vesicles (occupying >0.25 µm² in images) relative to the area occupied by the cell. (C) EIPA blocks ZEBOV infection. Vero cells were pre-treated with the indicated concentrations of EIPA, followed by incubation with gfpZEBOV (top panel) or rfpVSV (middle panel) each at MOI of 0.1 in the continued presence of the drug. Control cells received DMSO instead of the drug. After 24 h, cells were washed and fixed. Virus infection was determined by counting fluorescent foci. Cell monolayer integrity was confirmed by phase-contrast microscopy (bottom panel). (D) Quantitation of data shown in (C). Solid bars represent gfpZEBOV and open bars represent rfpVSV. Data were normalized to the average number of foci seen for untreated cells. Similar results were obtained when HEK293T cells were used (not shown). (E) EIPA-mediated block is at the entry step of infection. The mechanism of the EIPA-mediated inhibition of infection was examined by performing entry assays. HEK293T cells were pre-treated with the indicated concentrations of EIPA for 1 h followed by incubation with ZEBO-VLP (solid bars) or VSV-VLP (open bars) for an additional 3 h in the continued presence of the drug. Subsequently, cells were washed, and luciferase activity was measured for each sample. The results are expressed as luciferase activity relative to that in control (DMSO-treated) cells. The data represents average ± st.dev. of 3 independent experiments each performed in duplicate. (F) EIPA does not affect ZEBO-VLP binding to cells. HEK293 cells were pre-treated with EIPA (50 µM) for 1 h at 37°C, followed by incubation with ZEBO-VLPs for 10 min at room temperature. Cells were then washed to remove unbound virus, resuspended in luciferase assay buffer containing triton X-100 detergent, and luciferase activity was measured. Data were normalized to luciferase activity in vehicle-treated samples. Each data point represents mean ± st.dev. of 3 experiments. (G) EIPA treatment inhibits cellular uptake of ZEBO-VLPs. Vero cells were treated with DMSO or EIPA (50 µM) for 30 min. Subsequently, cells were incubated with gfpZEBO-VLP (green) in the presence of the inhibitor. After 30 min, cells were washed, fixed and the cell periphery was visualized by staining with phalloidin (red), staining cortical actin. Images were taken by confocal microscopy using 100× oil immersion objective lens. Only the mid-optical section representing the cell interior is shown. (H) VLP uptake was quantified by counting the total number of internalized VLPs in cells in each image (4–6 cells/image). A total of 10 images were analyzed for each sample. The data are presented as average number of VLPs per cell ± st.dev. (I) Internalized ZEBO-VLPs colocalize with dextran. HEK293T cells were incubated with gfpZEBO-VLP at 16°C. After 15 min, samples were washed and incubated with Alexafluor₅₉₄-labeled dextran 10,000 W (1 mg/ml) at 37°C. At indicated time intervals, cells were fixed and analyzed by confocal microscopy. Each image represents a mid optical section. Arrowheads indicate examples of association between gfpZEBO-VLPs (green) with dextran-containing vesicles (red). Nuclei (blue) were stained with DAPI. Quantitation of VLP colocalization (right panel). Multiple sections of each image were analyzed for VLPs that exhibited colocalization with dextran and their number expressed as percent of total VLPs in those sections. At least 10 images (5–6 cells/image) were analyzed for each sample. Mean ± st.dev. are shown. Note: this is likely an underestimate of the association as VLPs out of the plane or close to but not completely overlapping dextran positive vesicles were not counted. (J) ZEBOV induces dextran uptake by cells. Vero cells were incubated with replication-competent VSV or ZEBOV (MOI  = 5) for 15 min. Control cells were incubated with growth medium alone (none). Cells were then washed and incubated with medium containing fluorescently-labeled dextran 10,000 MW (1 mg/ml). After 30 min, cells were washed and fixed. Images were then taken by confocal microscopy using a 100× oil immersion objective lens, and the number of dextran-containing vesicles in individual cells were counted. For each sample, at least 10 images (≥25 cells) representing randomly selected fields were analyzed. The data represent mean ± st.dev. of dextran-containing vesicles/cell. A similar outcome was observed when HEK293T cells were incubated with ZEBO-VLP (not shown).