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Figure 1 | Retrovirology

Figure 1

From: Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum

Figure 1

Poly-ubiquitination of CD4 is required for Vpu-mediated CD4 degradation. A. HEK 293T cells were mock-transfected or co-transfected with 1.5 μg of SVCMV CD4 wt and 8 μg of SVCMV Vpu+ (Vpu+) or the phosphorylation-defective Vpu mutant SVCMV Vpu S52,56/N (Vpu S52,56/N). In parallel, CD4/Vpu transfectants were co-transfected with 8 μg of plasmids encoding his(6)/c-myc-Ub wt (myc-Ub wt) or the TDN mutant of ubiquitin his(6)/c-myc-Ub K48/R (myc-Ub K48/R). Transfected cells were treated with BFA, pulse-labeled with [35S]methionine and [35S]cysteine and chased with complete media for the indicated time intervals. Cells were then lysed and immunoprecipitated sequentially with anti-CD4 monoclonal and polyclonal antibodies first and then with anti-Vpu and anti-myc antibodies. B. Using quantitative scanning of CD4 bands from three independent experiments, the percentage of CD4 remaining over time as compared to time 0 is plotted for each transfection. C. HEK 293T cells were mock-transfected or co-transfected as described in A. Cell transfectants were treated for two hours with BFA prior to lysis. Steady state levels of CD4, actin and tagged ubiquitin were analysed by western-blot. D. Quantitative analysis from three independent experiments showing the level of CD4 relative to CD4 expressed with Vpu S52,56/N (arbitrarily set at 100%) for each transfectant.

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