Fig. 1.

Down-regulation and redistribution of surface HFE and its ligand TfR by HIV-1 Nef in cell lines. (A) Alignment of amino acid sequence of the cytoplasmic portion of HLA-A2 and HFE. Conserved residues are shown in red, and the YxxA motif is underlined. (B) HeLa-HFE cells were transiently transfected with Nef-GFP, stained for surface HFE, TfR, MHC I, or epidermal growth factor receptor (EGFR), and analyzed by flow cytometry. Results show that, as Nef expression increases (along the x axis), surface levels of HFE, TfR, and, to a lesser extent, MHC I decrease (y axis), whereas EGFR expression is unchanged. (C) Aliquots of 293 cells stably expressing HFE or HFE mutants, as indicated, were transiently transfected with Nef-GFP, and Nef-positive and Nef-negative cells were assayed for surface HFE and TfR. The results show that Nef-mediated down-regulation of HFE and TfR depends on a cytoplasmic tyrosine ³⁴²Yof HFE and correct folding of HFE (the C282Y mutation misfolds and does not bind TfR). (D-I) HeLa-HFE cells were transiently transfected with Nef-GFP, permeabilized, and stained for HFE (E and F, red) and for TGN46 (reveals the TGN) (H and I, red). Nef (D, F, G, and I, green) locates mostly to a perinuclear structure overlapping the TGN (I, yellow; nuclei stained blue with DAPI). HFE (E and F, red) locates to the cell membrane and endosomes in untransfected cells (E, arrow and F Left) but is redistributed to a perinuclear intracellular region overlapping Nef in transfected cells (E, arrowheads and F, yellow). wt, wild type. (Scale bar, 10 μm.)