Figure 1.

Effect of mutations in Nef from SIV_mac239 on its internalization. (A) Schematic representation of Nef from SIV_mac239 and location of different endocytic motifs. Whereas the two tyrosine-based endocytic motifs (Y₂₈GRL and Y₃₉SQS), which were implicated in the binding to AP-2, are found near the N terminus, the three motifs implicated in the binding to β-COP (D₁₈₄E₁₈₅), AP complexes (L₁₉₄M₁₉₅), and V1H (D₂₀₄D₂₀₅) are located in the C-terminal flexible loop of the protein. (m) is myristate. (B) Effects of mutations in Nef from SIV_mac239 on its internalization at steady state. Hybrid CD8-Nef proteins were expressed in Jurkat or 293-T cells and their surface levels [% CD8(+)] were evaluated by FACS. The surface expression of CD8 alone (lane 2) was set to 100% and the amount of internalization of the chimeras (lanes 3–11) was calculated relative to this value. The empty pEF-BOS vector was used as a negative control (lane 1). Error bars indicate SEM from at least three independent experiments. (C) Levels of expression of Nef chimeras in 293-T and Jurkat cells. Levels of different Nef proteins were determined by Western blotting (lanes 2–11). They were compared with the endogenous actin.