Steric Shielding of Surface Epitopes and Impaired Immune Recognition Induced by the Ebola Virus Glycoprotein
Figure 5
Surface N- and O- linked glycans contribute to GP-mediated shielding.
(A, left plot) Cells transfected with empty vector were mock incubated (grey shading) or incubated with neuraminidase and PNGaseF (black trace), stained for β1 integrin and assayed by flow cytometry. (A, right plot) Cells were treated with DMSO, transfected with empty vector, and mock incubated (grey shading) or treated with benzyl-α-GalNAc (bz-GalNAc), transfected with empty vector, and incubated with PNGaseF (black trace), then stained for β1 integrin and assayed by flow cytometry. (B) Cells were left untreated and untransfected (grey shading), or treated with bzGalNAc, transfected with vector encoding GP, and incubated with PNGaseF (black trace), then assayed by flow cytometry for the internal proteins GM130 and calnexin to show the effect of these treatments on cell permeabilization. As a positive control, bz-GalNAc-treated, fixed/permeabilized cells are shown (dashed trace). (C, D) Floating cells in cultures transfected with vector encoding GP were mock incubated or incubated with neuraminidase and/or PNGaseF, then analyzed by Western blot with rabbit polyclonal antibodies to GP (C, left blot) or stained for β1 integrin and assayed by flow cytometry (D). (C, E) Cells were treated with DMSO or bz-GalNAc, then transfected with empty vector, or vector encoding GP. Cells were then mock-incubated, or incubated with PNGaseF, then analyzed by Western blot (C, right blot) or stained for β1 integrin and assayed by flow cytometry (E). For D and E, mock-incubated cells or DMSO-treated cells transfected with empty vector = grey shading; GP-transfected and DMSO-treated or mock-incubated cells = black traces; GP-transfected and bz-GalNAc- and glycosidase-treated cells = dashed traces. In Western blot panels (C), selected samples were lysed and denatured before incubation with PNGaseF for comparison, as indicated.