Figure 1. Clathrin and caveolar endocytosis are not required for ZEBOV entry.

(A) Regulators of clathrin and caveolae-mediated endocytosis are not important for ZEBOV infection. The role of proteins important for endocytosis in ZEBOV infection was assessed using dominant negative (DN) effector proteins. HEK293T cells were transfected with plasmids encoding GFP, DN-Eps15-GFP or DN-Cav1-GFP. Twenty-four h post-transfection cells were inoculated with wild-type ZEBOV (MOI  = 0.2). Cells were fixed 48 h later, stained for nuclei using DAPI and for ZEBOV VP40 matrix protein using a specific rabbit antiserum followed by Alexa₆₃₃ secondary antibody. Images were taken by fluorescence microscopy and analyzed as described in the methods. To quantitate the infection dependency of ZEBOV on expression of each construct, 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. The data were averaged for all replicates (>5) and normalized to that seen in cells transfected with GFP alone. (B) To measure the impact of expression of each GFP-tagged protein on the virus entry step into cells, a contents mixing assay was performed. Cells were transfected as above and then used in the assay 36 h after transfection. Both ZEBO-VLPs and VSV-G pseudotyped particles were used as indicated. Measurements were made at 3 h, at which time the contents mixing signal peaked in untreated cells (peak is at 2–3 h post cell binding). Measurements were normalized to untransfected cells. The results are mean ± st.dev. of 3 independent experiments. (C) To test DN-Cav1 efficacy, HEK293 cells were transfected with plasmids encoding GFP or DN-Cav1 tagged with GFP. Thirty six hours after transfection cells were infected with a recombinant 10A1 MLV virus encoding a truncated CD4 receptor as a marker for infection. 36 h after the infection cells were stained for CD4 expression with anti-CD4 antibody conjugated to PE (red) and cells expressing CD4 and the GFP-tagged protein by microscopy. Data were analyzed as described in the methods and in (A). (D) Cholera toxin B subunit uptake is blocked in cells expressing DN-Cav-1. As an additional test of DN-Cav-1 efficacy, the impact of expression on cholera toxin subunit B (CTxB) uptake was measured. HEK293T cells were transfected with plasmid encoding GFP (left panel) or GFP-tagged DN-Cav1 protein (right panel). Thirty-six h after transfection cells were incubated with fluorescently-labeled CTxB for 30 or 60 min, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Green  =  GFP or DN-Cav1; Red  =  CTxB. (E) ZEBO-VLPs do not associate with markers of caveolae or clathrin-coated endosomes. Vero cells were preincubated with gfpZEBO-VLPs at 16°C (to prevent endocytosis) for 15 min to allow virus attachment. Excess virus was then removed and the temperature raised to 37°C (to initiate endocytosis) prior to fixation at indicated times. For caveolin-1 and clathrin light chain A, permeabilized cells were stained with anti-Cav1 antibody or anti-CLCA antibody followed by Alexafluor₅₉₄-conjugated secondary antibody. For transferrin, Alexafluor₅₉₄-labeled transferrin was added to cells during incubation with the VLPs. DAPI was used to stain nuclei (blue). Images were taken by confocal microscopy with a mid z-section shown. Green  =  gfpZEBO-VLPs; Red  =  indicated endocytic marker.