Fig. 4.

Effects of mutations in Nef on Nef-mediated down-regulation of HFE, TfR, and MHC I. (A-D) HeLa-HFE cells were transfected with Nef-GFP constructs (x axis) encoding wild-type Nef (A), ⁶²EEEE⁶⁵/AAAA Nef (B), ²⁰M/A Nef (C), and ⁷²PxxP⁷⁵/AxxA Nef (D) and stained for surface HFE (Top, y axis), TfR (Middle, y axis), and MHC I (Bottom, y axis). The results show that the ability of Nef to down-regulate HFE and TfR is retained by Nef variants lacking ⁶²EEEE⁶⁵ or ²⁰M, but the ⁷²PxxP⁷⁵ motif is required for HFE/TfR down-regulation. All three motifs are needed for down-regulation of MHC I. (E) The role of other Nef motifs. HeLa-HFE cells were transfected with the Nef-GFP variants indicated and stained for surface MHC I, HFE, and TfR. The percentage of down-regulation, relative to down-regulation by wild-type Nef-GFP (100%), was calculated. Myristolation and the proline-rich domain are required for MHC I, HFE, and TfR down-regulation; four Nef motifs, needed for CD4 down-regulation, are dispensable.