FIG. 1.

Nef expression slows down the internalization of the Ii chain in a dileucine-dependent manner. (A) HeLa-CIITA cells were transfected with the indicated bicistronic plasmid, Nef NL4-3 or Nef NA7 (expressing Nef and GFP), or Nef-STOP (negative control expressing GFP only), collected 24 h later, stained for surface Ii chain, and analyzed by flow cytometry. (Left panels) Representative dot plot analyses are shown. (Right panel) The Nef-induced upregulation of the Ii chain on GFP⁺ cells was quantified, and the results were normalized using the Nef-STOP control as a reference. Data from four experiments were used to calculate means and their standard errors. WT, wild type. (B and C) Kinetics of internalization of Tf and the Ii chain. HeLa-CIITA cells were transfected with the indicated bicistronic plasmid, Nef NL4-3 or Nef NA7, or Nef-STOP. After 24 h, cells were collected and allowed to bind at 0°C with either the Ii chain-specific PE-labeled MAb or Alexa Fluor 647-labeled Tf. Cells were then shifted to 37°C for the lengths of time indicated. Ligands remaining on the cell surface were released with an acid wash of half of each fraction. Flow cytometry analysis of the cells allowed the measurement of the MFI on GFP⁺ cells. For each sample, total MFI and acid wash-resistant MFI were used to determine the percentage of internalized ligand. Data presented are representative of at least three independent experiments.