Multilayered Mechanism of CD4 Downregulation by HIV-1 Vpu Involving Distinct ER Retention and ERAD Targeting Steps
Figure 3
ER retention of CD4 mediated by Vpu.
(A–L) HeLa cells were treated without (mock) (A–H) or with siRNAs to NPL4 (I–L). Cells were then transfected with plasmids encoding human CD4 without (A–D) or with Vpu (E–L). At 12 h after transfection, cells were fixed, permeabilized and labeled with a rabbit polyclonal antibody to Vpu (green channel; A, E, I), IgG2a mouse monoclonal antibody to CD4 (red channel; B, F, J) and IgG1 mouse monoclonal antibody to calnexin (blue channel; C, G, K). Stained cells were examined by confocal fluorescence microscopy. Bars: 10 µm. (M–O) HeLa cells expressing Vpu were fixed and processed for immunoelectron microscopy as previously described [14]. Notice that enhanced nanogold particles indicative of Vpu are mainly associated with the ER and nuclear envelope (NE). In contrast, untransfected cells show no enhanced nanogold, confirming the specificity of the staining (Fig. S3). N: nucleus; PM: plasma membrane. Bars: 1 µm. (P) Total lysates from HeLa cells treated as in A–L were digested with Endo H, PNGase F or left untreated before immunoblotting with an antibody to the CD4 ectodomain. CHO: N-linked carbohydrate chain. (Q) Data are represented as mean ± SEM from three independent experiments like that in P. (R) HeLa cells treated as in A–L were analyzed for cell surface CD4 by FACS. (S) Bar graphs represent percentage of surface CD4 levels in cells expressing Vpu relative to CD4-surface levels in the absence of Vpu (100%). Values are expressed as mean ± SEM from three independent experiments.