Figure 3

Effect of Vpu on CD4 molecules lacking lysine residues in the cytoplasmic tail. A. Analysis of CD4 wt and CD4 KRcyto turnover in presence or absence of functional Vpu by pulse-chase labeling and immunoprecipitation. HEK 293T cells were mock-transfected or co-transfected with 2 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 20 μg of provirus encoding Vpu⁺ (HxBH10-vpu⁺) or phosphorylation-defective Vpu mutant (HxBH10-vpu S52,56/D). Cells were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine and chased in complete medium for the indicated time intervals. Cells were then lysed and immunoprecipitated sequentially with anti-CD4 antibodies first (polyclonal and monoclonal) and then with anti-Vpu antibodies. B. Using quantitative scanning of CD4 bands from two independent experiments, the percentage of CD4 remaining over time as compared to time 0 is plotted for each transfection. C. Effect of Vpu on steady-state CD4 wt and CD4 KRcyto levels. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt or pHIV CD4 KRcyto and 10 μg of proviruses encoding Vpu^- or Vpu⁺ in addition to 25 μg of the his(6)/c-myc-Ub K48/R expressor. In the left panel (Env^-), a similar experiment was performed except that HEK 293T cells were co-transfected with 10 μg of envelope-defective provirus (HxBc2-pr^-, vpu^-, env^- or HxBH10-pr^-, vpu⁺, env^-) and treated with BFA for 2 h prior to lysis. Cell lysates were then treated as described in the materials and methods section. D. Quantitative analysis of steady-state CD4 levels. CD4 levels in presence of absence of his(6)/c-myc-Ub K48/R were arbitrarily set at 100%. The levels of CD4 in presence of Vpu are shown relative to the corresponding controls. These results are representative of the data obtained in three independent experiments for Env^- and five independent experiments for Env⁺.