FIG. 5.

Degradation of CD4 in Nef-expressing cells. (A) HeLa-CD4 cells were cotransfected with a construct encoding GFP along with the indicated constructs. Cells were then sorted by FACS based on GFP expression and solubilized to analyze the expression levels of CD4, Nef, p56^lck, and GFP in cleared lysates. (B) CD4 levels were normalized to that of GFP and set to 100% in cells expressing neither Nef nor p56^lck (left). CD4 degradation calculated as (CD4_{no Nef} − CD4_Nef)/CD4_{no Nef} was set to 100% in cells expressing no p56^lck (right). An average of three independent experiments is shown (t test: *, P of <0.05; **, P of <0.01). (C) The impact of a 6-hour treatment with NH₄Cl (100 mM) and MG132 (20 μM) on the levels of cell surface CD4 (left) and the rate of CD4 internalization (right) was measured, as described in the legend to Fig. 1. (D) HeLa-CD4 cells were incubated with VSV-G-pseudotyped viruses expressing or not expressing Nef (+ and −, respectively) to achieve 100% of transduction efficiency. Cells were then treated for 6 h with the indicated chemicals and solubilized to analyze the expression level of CD4 (bottom) and p24 (top). CD4 levels were normalized to that of p24, and CD4 degradation was calculated for each treatment as 100 × (CD4_{no Nef} − CD4_Nef)/CD4_{no Nef}. Immunodetection signals were acquired as indicated in Materials and Methods. Error bars represent 1 standard deviation from the mean.