Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein
Figure 4
Removal of the GP1 subunit from the cell surface results in exposure of previously occluded surface epitopes.
(A-C) 293T cells were transfected with empty vector or vector encoding wt GP. Floating and adherent cells were harvested 24 h after transfection, pooled, and either left untreated, or incubated at 37°C for 20 minutes in 150 mM DTT. (A) Western blot analysis of the GP1 subunit shed into the supernatant of untreated or DTT-treated cells. (B) Cells were transfected with empty vector and assayed by flow cytometry to show baseline differences in surface staining for β1 integrin and MHC1 between untreated cells (grey shading) and DTT-treated cells (black trace). (C) Cells were transfected with vector encoding wt GP and assayed by flow cytometry for the internal proteins GM130 and calnexin to show the effect of DTT on cell permeabilization. Untreated cells are shown in the grey shading; DTT-treated cells are shown in the black trace, and, as a positive control, fixed/permeabilized cells are shown in the dashed trace. (D) Cells were transfected with vector encoding wt GP, were mock- or DTT-treated, stained for GP and β1 integrin or MHC1 as described above, and assayed by flow cytometry. (E, F) 293T cells were transfected with empty vector or vector encoding wt GP or GP cl(-). Cells were harvested and treated as above. (E) Western blot analysis using rabbit polyclonal antibodies of GP1 or GP0 shed into the supernatant of untreated or DTT-treated cells. (F) Transfected and treated cells were surface stained for MHC1 and assayed by flow cytometry. Empty vector-transfected, untreated cells are shown in the grey shading; GP-expressing cells are shown after mock treatment (black trace) or DTT treatment (dashed trace).