Supplementary Figure 1. Impact of increasing amounts of p56lck on Nef-induced CD4 down-regulation. A) HeLa-CD4 cells were transfected with 2 µg of a Nef-expressing plasmid (WTNef, white bars) or pUC19 (Mock, black bars) and increasing amounts of a p56lck-expressing plasmid (2, 4 or 6 µg). Cell-surface expression of CD4 was analyzed at steady state by flow cytometry. Results are normalized to cell-surface CD4 expressed on mock-transfected cells and expressed as percentages. B) CD4 down-regulation activity of Nef. Results are normalized to the efficiency of CD4 down-regulation measured in the absence of p56lck and expressed as percentages. Representative results from two independent experiments are shown.
Supplementary Figure 2. Effect of a 6-hour treatment with NH4Cl and MG132 on lysosome acidification and proteasome activity. The effect of NH4Cl and MG132 was measured on productive infection of HeLa-CD4 cells with a VSV-G pseudotyped GFP-reporter virus (see Figure 3 legend). A viral dilution allowing infection of ~30% of mock treated target cells was used. Cells were treated with 100 mM NH4Cl or 20 µM MG132 for 6 h either during infection or 16 h post infection. Cells were harvested 60 h post infection and the percentage of GFP-positive cells was recorded. The percentage of infected cells normalized to that of mock treated cells is ploted.
Supplementary Figure 3. Contribution of HIV-1 Nef motifs to CD4 downregulation. HPB-ALL were transfected with plasmids expressing WT or mutant Nef-GFP fusions (NefLL/AA-GFP, NefDD/AA-GFP and NefPxxP/AxxA-GFP). Transfected cells were gated on the basis of GFP expression. Cell-surface expression levels of CD4 (A) and the rate of CD4 internalization (B) were measured as described in Fig. 1 legend.
Supplementary Figure 4.HIV-1 and SIVmac239 Nef do not cause an increase of CD4 internalisation rate in myeloid cells. THP-1 cells were transfected with plasmids expressing HIV-1 or SIVmac239 Nef in combination with a GFP encoding plasmid. Transfected cells were gated on the basis of GFP expression. Cell-surface expression levels of CD4 (A) and the rate of CD4 internalization (B) were measured as described in Fig. 1.
Supplementary Figure 5. Similar expression levels of Nef cause an increase of CD4 internalization in HPB-ALL but neither in THP-1 nor in HeLa-CD4. HPB-ALL, THP-1 and HeLa-CD4 cells were transfected with a Nef-GFP or GFP expressing plasmid.Cells were gated based on GFP fluorescence and populations with similar Nef-GFP or GFP expression levels were selected (A), numbers indicate the mean fluorescence intensity of GFP in the selected populations. The rate of CD4 internalization (B) was measured as described in Fig. 4C legend within the population selected in (A) where Nef-GFP and GFP expression is similar for all three cell lines. Flow cytometry profiles in (A) are representative of 3 independent experiments. Values in (B) are the means of three independent experiments; error bars represent 1 SD from the mean.
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