FIG. 4.

Nef binds HLA-A2 but not HLA-E. (A) Amino acid alignment of the cytoplasmic tails of the murine MHC-I molecule H2-K^d and the human MHC-I molecules HLA-A2 and HLA-E. Amino acids that are important for downmodulation of MHC-I by Nef are highlighted (4, 22). Shaded residues indicate the differences between HLA-A2 and HLA-E. (B) The HLA-A2 cytoplasmic tail is necessary for Nef-mediated downmodulation. Astrocytic cell lines expressing the indicated MHC-I molecule were transduced with adeno-Nef or control-adeno at an estimated MOI of 100:1. After 24 h, the cells were stained with an antibody against H2-K^d and analyzed by flow cytometry. Fold downmodulation was calculated by comparing the mean fluorescence intensity of cells transduced with control-adeno to that of those transduced with adeno-Nef (nontransduced cells had fluorescence similar to that of control-adeno-transduced cells). (C) The HLA-A2 cytoplasmic tail is necessary for Nef binding. Astrocytic cells stably expressing the indicated MHC-I molecule were transduced with adeno-Nef at an estimated MOI of 100:1. They were metabolically labeled for 4 h in the presence of 25 mM NH₄Cl, treated with DSP, and immunoprecipitated using an antibody against H2-K^d or mouse IgG. Results shown are representative of six independent experiments. (D) Mutation of Y₃₂₀ disrupts Nef binding. Coimmunoprecipitation experiments were performed as described for panel C, using astrocytic cells stably expressing MHC-I molecule H2-K^d, H2-K^d/A2, or H2-K^d/A2 Y₃₂₀A. The results are representative of three independent experiments. (E) Quantitation of Nef binding. The percentage of MHC-I molecules bound to Nef was quantitated as follows: the number of Nef counts specifically coprecipitating with MHC-I was determined using a phosphorimager. Nef counts were then corrected for the different numbers of methionines and cysteines in Nef versus MHC-I (7 versus 10 to 14 depending upon the chimera). Assuming a 1:1 ratio of Nef to MHC-I molecules, the percentage of MHC-I molecules bound to Nef was determined by dividing the counts from bound MHC-I molecules (equivalent to corrected Nef counts) by the total MHC-I counts × 100. Results shown are means ± standard deviations from six independent experiments.