Figure 2

Effect of Vpu on CD4 ubiquitination. A. Vpu-mediated ubiquitination of CD4 wt when CD4 is retained in the ER through treatment with BFA. HEK 293T cells were mock-transfected or co-transfected with 1 μg of SVCMV CD4 wt, 8 μg of SVCMV Vpu⁺ or the phosphorylation-defective Vpu mutant SVCMV Vpu S52,56/N and 8 μg of the TDN mutant his(6)/c-myc-Ub K48/R. Samples were then treated as described in the materials and methods section. CD4 molecules were immunoprecipitated with anti-CD4 polyclonal antibodies prior to western-blot analysis with anti-myc monoclonal antibodies. (triangle) indicates the position of the heavy chains of anti-CD4 antibodies. B. Quantitative analysis of ubiquitinated CD4 conjugates. (asterisk) represents the area of the autoradiogram that was used for quantitation of CD4-Ub conjugates. The histogram shows the relative levels of ubiquitinated CD4 conjugates in presence or absence of a functional Vpu. Relative CD4-Ub conjugate levels were evaluated by quantitation of the signal detected in the area delineated on the autoradiogram relative to total CD4 as determined by quantitation of the band detected with the anti-CD4 antibodies on whole cell lysate. The relative level of ubiquitinated CD4 detected in absence of Vpu was arbitrarily set at 1. The data represent results from seven experiments. C. Vpu-mediated ubiquitination of CD4 wt in condition where CD4 is retained in the ER through binding with HIV-1 Env. HEK 293T cells were mock-transfected or co-transfected with 1 μg of pHIV CD4 wt, 10 μg of provirus encoding Vpu^- (HxBH10-vpu^-) or Vpu⁺ (HxBH10-vpu⁺) and 20 μg of his(6)/c-myc-Ub K48/R. Samples were then treated as in A but in absence of BFA. D. Quantitative analysis showing the relative levels of ubiquitinated CD4 detected in two independent experiments. Relative levels of ubiquitinated CD4 conjugates were determined as described in B.