Figure 3

Inhibition of CME with a DN form of Eps15 significantly reduces ABLV G-mediated viral entry into HEK293T cells. HEK293T cells transfected with an eGFP control plasmid or eGFP-tagged Eps15 mutants were infected with VSVΔG-ABLV G* or –VSV G*-RFP pseudoviruses at a MOI = 2 for 20 hrs and then analyzed for RFP expression as described in Figure 1. Under these conditions, a MOI of 2 yielded 25-30% virus-infected cells among cells transfected with the eGFP control plasmid. DIIIΔ2, a mutant of Eps15 that does not affect clathrin coated pit formation; EH29, DN Eps15 mutant. (A) Representative microscopic fields of cells transfected with each Eps15 construct and infected with VSVΔG-ABLVp G*- RFP pseudovirus. Similar results were obtained for ABLVs G-expressing pseudovirus (not shown). (B) Quantitation of virus-infected cells. A minimum of 1000 cells were counted for each sample per experiment. VSVΔG- VSV G* was included as positive control. Results are expressed as percent virus-infected cells relative to that of controls and represent 3 independent experiments; error bars are SEM. Significance of effect on virus infection was determined using Student’s t-test. **, p < 0.0005; *, p < 0.005. (C) Cholera toxin B (CTX-B) subunit uptake into HEK293T cells is not inhibited by EH29. To verify the specificity of EH29 to inhibit CME, 18 hrs post transfection HEK293T cell monolayers grown on 12 mm coverslips were incubated with Alexa Fluor 594-labeled CTX-B (10 μg/ml) for 1 hr at 37°C. Cells were then washed twice with PBS, fixed and imaged. Images were taken by confocal microscopy with a mid z-section shown. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride).