An N-terminal AP2 binding motif is required and sufficient for upregulation of TfR by Nef. (A) PBMC were infected with HIV-1 coexpressing GFP and various mutants of the SIVmac 239 Nef protein and assessed for TfR, CD4, CD28, CD3 and MHCI modulation by FACS 48 hours post infection. The table shows the qualitative activity of the Nef mutants in PBMC and the subsequent experiment of transfected 293 T cells with pCG-IRES-GFP expression plasmids of a truncated 239 Nef protein in combination with a variety of in-frame deletions. By this approach we could narrow down the critical domain for Nef-mediated TfR modulation to a four amino-acid deletion in the flexible N-terminal loop (Δ26-29). (B) PBMC were infected with HIV-1 NIG expressing the NL4-3 Nef or a variant in which we reconstituted the YGRL motif at position 28. In addition we infected PBMC with the HIV-1 NIG version of SIVmac 239 Nef and a mutant harbouring a change of Y28 to F. At 48 hours post infection modulation of TfR, CD4, CD28, CD3 and MHCI was assessed by flow cytometry. The graph shows mean values and SD of three independent infection experiments. For TfR modulation we have included the respective MFI of the infected GFP + cell population in the primary FACS plots.