FIG. 5.

Effect of prototypic sorting signals and Nef on the recycling of HLA-A2. HeLa cells were transfected with 4 μg of the indicated A2 vectors and 0.5 μg of GFP vector in the absence (left panel) or in the presence of 12 μg of Nef-FT expression vector. After 20 h, cells were pretreated for 2 to 3 h with cycloheximide (100 μg/ml) to eliminate de novo synthesis of MHC-I molecules. An aliquot of the cells was stained with the BB7.2 MAb to define HLA-A2 steady-state surface levels. To disrupt cell surface MHC-I complexes, the remaining cells were exposed to an acidic buffer that removed surface β2-microglobulin. Cells were then incubated at 37°C for the indicated periods of time, and the surface expression of HLA-A2, from a preexisting intracellular pool, was measured by flow cytometry in GFP-positive cells after staining with the BB7.2 MAb. Steady-state surface levels were defined as 100%, and the fluorescence intensity at time zero, after the acidic wash, was defined as 0%. The data show the ratio of the mean fluorescence at different time points to the value obtained for steady-state levels. In a typical experiment, steady-state levels were 750, 690, and 80 U for A2 WT, A2 ASQA, and A2 YSQI without Nef and 210, 680, and 50 U with Nef, respectively. Results from three independent experiments (mean ± standard deviation) are shown.