FIG. 5.
FACS analysis of CD4 downregulation by wild-type and mutant Nef proteins. HeLa cells were cotransfected with pCMV.CD4 and either wild-type or mutant versions of pNL4-3 Nef.IRES.GFP, which is designed to express Nef and GFP as separate proteins from the same bicistronic transcript. For the FACS analyses, GFP fluorescence was used as a marker to identify cells that had been transfected with a Nef expression plasmid. (A) FACS histograms of CD4 surface levels in cells transfected with the plasmids mentioned above. For each condition, the relative amount of CD4 at the plasma membrane is shown for cells transfected with the pIRES2.GFP empty vector (no Nef expression) (thin lines) and for cells transfected with wild-type or mutant Nef (bold lines). The fluorescence profile of cells left untransfected (no CD4 expression) (solid gray shading) is also shown. (B) (Top) Immunoblot (IB) analysis of the expression of wild-type and mutant versions of Nef by using an antiserum to HIV-1 Nef. Cells transfected with the empty vector pIRES2.GFP (no Nef expression) were used as a control. (Bottom) Quantification of the results in panel A. Bars represent geometric means ± standard errors of the means from three independent experiments. Levels of CD4 on the surfaces of Nef-expressing cells are compared to those on the surfaces of cells transfected with the pIRES2.GFP empty vector (taken as 1). (C) FACS analysis of CD4 endocytosis in cells expressing wild-type or mutant versions of Nef. Data for cells that were transfected with the empty vector and do not express Nef are included as a control.