Initial characterization of the E, N, T, and S mutants: protein expression, modulation of CD4, surface expression of CD8 chimeras, and modulation of class I MHC. (A) Immunoblot analysis of Nef protein expression. Cells (HEK 293T) were transfected with pCG-based plasmids (0.5 μg) expressing wild-type Nef-GFP (wt) or the indicated missense mutants and then analyzed for Nef expression by Western blotting; simultaneous detection of tubulin was used as loading control. Ctrl, cells transfected with pCG-GFP. (B) Nef-mediated down-regulation of CD4. Cells (HEK 293T) were transfected with 0.5 μg pCMX-CD4; 0.03, 0.1, or 0.3 μg pCG-Nef-GFP or the pCG-Nef-GFP variants; and pCG-GFP as required to a total of 0.3 μg of GFP expression construct. One day later, the cells were stained for CD4 with an Alexa 647-conjugated antibody and analyzed by two-color flow cytometry. The mean Alexa 647 (CD4) fluorescence intensity of the GFP-positive cells is graphed. (C) Expression of CD8-Nef chimeras on the cell surface. Cells (HEK 293T) were transfected with plasmids expressing CD8-Nef chimeras in which the extracellular and transmembrane domains of the CD8 α chain are fused to Nef as the cytoplasmic domain. The cells were stained for CD8 using a fluorescein isothiocyanate-conjugated antibody at 4°C in the presence of azide, fixed, and then analyzed by flow cytometry. The mean fluorescence intensity of the cells is graphed; error bars are the standard deviation of the mean for duplicate transfections. (D) Nef-mediated modulation of class I MHC. Cells (HeLa) were transfected with the Nef-GFP expression plasmids described above (1 μg). One day later, the cells were stained with a phycoerythrin-conjugated antibody to HLA-A, -B, and -C and analyzed by two-color flow cytometry. The mean PE (class I MHC) fluorescence intensity of the GFP-positive cells was used to calculate activity relative to wild-type Nef; error bars are the standard deviation of the mean for duplicate transfections.

Up-regulation of DC-SIGN to the cell surface. Cells (HeLa) were cotransfected with pCG-based plasmids expressing GFP, wild-type Nef-GFP, or the indicated Nef-GFP mutants, along with a plasmid expressing DC-SIGN (0.5 μg). One day later, the cells were stained for DC-SIGN with a phycoerythrin-conjugated antibody and analyzed by two-color flow cytometry. (A) Representative histograms of relative cell number versus PE fluorescence intensity for the GFP-positive cells; the percentage of positive cells is indicated. (B) The relative activity of each Nef mutant for the up-regulation of DC-SIGN. The mean fluorescence (PE) intensities for cells expressing the Nef-GFP mutants are expressed as percentages relative to that of cells expressing GFP (set to 0%) and cells expressing wild-type Nef-GFP (set to 100%). Values are the means of three independent transfections; error bars are the standard deviation from the mean. (C). Relative expression of Nef-GFP and the mutants; lysates of the cells analyzed in panel A were analyzed by Western blotting using an antibody to GFP.

Pull-down of intact AP-1 or AP-3 from cytoplasmic lysates by GST-Nef. Lysates of HeLa cells were incubated with equal amounts of purified wild-type or mutated GST-Nef, previously immobilized on glutathione-Sepharose beads. Bound proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the association of AP complexes was analyzed by Western blotting with anti-γ-adaptin (panel A) or anti-δ-adaptin (panel B). Unfractionated cell lysates were run as a control for the detection of γ- and δ-adaptin (right lanes, input). The bands were quantified using NIH Image software and the values indicated are expressed as the percentage of binding relative to that of wild-type Nef. In panel (C), the Coomassie blue-stained gel indicates the relative loading of GST fusion proteins on the beads.

Nef-mediated stabilization of AP-1 on endosomal membranes. HeLa cells (clone P4.R5) were transfected with plasmids expressing CD8-Nef chimeras. One day later, the cells were treated with 10 μg /ml brefeldin A (BFA) for 5 min at 37°C and then fixed, permeabilized, and stained for γ-adaptin (AP-1) by indirect immunofluorescence with anti-γ-adaptin and rhodamine X-conjugated secondary antibody (red) and for CD8 by direct immunofluorescence with a fluorescein isothiocyanate-conjugated anti-CD8 antibody (green). For each field, a Z-series of images was acquired at 0.5-μm steps using an Olympus disk-scanning confocal microscope; the images were processed using no-neighbor deconvolution (Slidebook software); and the image shown is a projection of the Z-stack.

Up-regulation of the invariant chain (Ii) of class II MHC to the cell surface. (A) HeLa-CIITA cells were cotransfected with pCG-GFP (0.1 μg) and pCIneo-derived plasmids expressing either native, wild-type Nef or the indicated Nef mutants (4 μg). After 2 days, the cells were stained for Ii with a PE-conjugated antibody and analyzed by two-color flow cytometry. Neo, cells cotransfected with the empty plasmid pCIneo. Representative histograms of relative cell number versus PE (Ii) fluorescence intensity for the GFP-positive cells are shown; the percentage of Ii-positive cells is indicated. (B) Relative levels of surface Ii in the transfected cells. The mean PE (Ii) fluorescence intensities (MFI) for the GFP-positive cells are expressed relative to cells expressing wild-type Nef. Wild-type (WT) Nef was set at 100%; the mean fluorescence intensity of the Nef-negative control is the actual value relative to that of the wild type (WT) and was not normalized to zero. Values are the means of two independent transfections; error bars are the standard deviation from the mean. (C) Phenotype of the E160K mutant as a fusion with GFP. HeLa-CIITA cells were transfected with pCG-based plasmids (4 μg) expressing wild-type Nef-GFP, the indicated missense mutants, or no Nef (“GFP”) and then stained for Ii 2 days later with PE-conjugated antibody and analyzed by two-color flow cytometry. The mean PE (Ii) fluorescence intensities for the GFP-positive cells are expressed relative to that of cells expressing wild-type Nef. Wild-type Nef (Nef-GFP) was set at 100%; the mean fluorescence intensity of the Nef-negative control (GFP) is the actual value relative to that of Nef-GFP and was not normalized to zero. Values are the mean of two independent transfections; error bars are the standard deviation from the mean. (D) Aliquots of the same populations of cells analyzed in panel A were analyzed for Nef expression by Western blotting; simultaneous detection of tubulin was used as a loading control. (E) Dose-response curve of Ii up-regulation using native Nef. The experiment was performed as in panels A and B, except that the amount of pCIneo-based plasmid expressing wild-type Nef was varied as shown; all transfections were normalized to 4 μg total of pCIneo-based plasmid using the empty pCIneo.

Nef-mediated modulation of transferrin trafficking. (A) Transferrin uptake. HeLa cells were transfected with plasmids expressing GFP, wild-type Nef-GFP, or the indicated Nef-GFP mutants. Twenty-four hours later, the cells were incubated for 2 h in serum-free medium at 37°C to deplete endogenous transferrin and then exposed to 10 μg /ml Alexa 594-conjugated transferrin in the same medium at 37°C for 30 min to allow uptake. The cells were then fixed and analyzed by confocal microscopy using a Bio-Rad MRC1000 microscope. The images shown are representative median optical sections. Asterisks indicate cells expressing any detectable Nef-GFP. (B) Down-regulation of transferrin receptor. HeLa cells were transfected with pCG-based expressing Nef-GFP or the indicated mutants (1 μg). One day later, the cells were stained for transferrin receptor with a phycoerythrin-conjugated antibody and analyzed by two-color flow cytometry. Using the mean PE (transferrin receptor) fluorescence intensity of the GFP-positive cells, the activity of each mutant in down-regulating transferrin receptor was calculated relative to that of wild-type Nef, which was set at 100%.

Binding of Nef to AP-1 and AP-3 hemicomplexes detected by yeast three-hybrid assay. Yeast cells were cotransformed with two plasmids: one plasmid expressed Nef as a fusion with the GAL4 DNA binding domain together with the σ subunit of either AP-1 (A) or AP-3 (B), and the second plasmid expressed GAL4 activation domain fusions with either the γ subunit of AP-1 (A) or the δ subunit of AP-3 (B). Colonies of cotransformed yeast cells pooled, patch-plated on nonselective (+His) medium and then replica plated onto nonselective (+His) and selective (−His) medium. Growth over background on −His medium indicates a protein-protein interaction. Empty, yeast cotransformed with a vector expressing no Nef and no σ subunit, together with the vector expressing the γ-GAL4 activation domain fusion (A) or the δ-GAL4 activation domain fusion (B). Wild-type, native Nef, sequence ENTSLL. The mutants are indicated at the left.

Nef-mediated stabilization of AP-3 on endosomal membranes. HeLa cells (clone P4.R5) were transfected with plasmids expressing the CD8-Nef chimeras, treated with brefeldin A, and imaged exactly as described in the legend to Fig. 7, except that the indirect immunofluorescence was done using a primary antibody to the δ subunit of AP-3.