FIG 3.
HIV-1 Vpu downmodulates HLA-C without changing total HLA-C protein levels. (A) PBMCs or purified CD4+ T cells were infected with the indicated wild-type (wt) or vpu-defective (vpu−) HIV-1 clones. At 3 days postinfection, total HLA-C protein levels were determined by permeabilization, staining, and subsequent flow cytometric analysis. Representative primary data are shown on the left. (B) HEK293T cells were cotransfected with the indicated molecular clones of wt or vpu-defective HIV-1 and an expression plasmid for V5-tagged HLA-C. All transfections except for wt and vpu-defective STCO were harvested at 2 days posttransfection for Western blot analyses. Cells transfected with wt and vpu-defective STCO were harvested at 3 days posttransfection to increase Vpu expression levels. One representative Western blot out of three is shown on the left. HLA-C protein levels were quantified and normalized to the GAPDH level. Wild-type-infected samples were set to 100% (right). (C) Cells were transfected as described above for panel B, and HLA-C protein levels at the cell surface were determined by flow cytometry at 2 days posttransfection. Bar diagrams show mean values from three to six independent experiments ± SEM. Asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.01).