Figure 3

HIV-1 Gag attenuates SDF-induced CXCR4 downregulation in Jurkat T cells. (A) Jurkat T cells were pre-treated with cycloheximide, then incubated in the presence (filled squares) or absence (open squares) of SDF, PMA and ionomycin for the indicated times. At each time point, cells were lysed and analysed by SDS-PAGE and Western blotting with an anti-CXCR4 polyclonal antibody. Western blots were quantitated and amount of CXCR4 remaining at each time point was determined as a percent of amount of CXCR4 at 0 hour. Data from one representative experiment (out of three) is shown. (B) Jurkat T cells were transduced with Gag-GFP encoding lentiviruses at the indicated MOIs. 48 hours post transduction, cells were analyzed for GFP fluorescence by flow cytometry. The % of GFP positive cells is indicated for each MOI. (C) A representative gel depicting CXCR4 levels in Jurkat T cells transduced with lentiviruses encoding wild-type Gag-GFP and treated as described in (A) is shown. Control represents untransduced cells. (D) Quantitation of the gel shown in (C). Additionally, degradation of CXCR4 in Jurkat T cells transduced with lentiviruses encoding LTAL Gag-GFP is shown. Error bars represent standard deviation between duplicates at each time point. (E) Jurkat T cells were transduced with lentivirus encoding LacZ (control), Gag-GFP or LTAL Gag-GFP. Data shown represents the mean ± SD of % undegraded CXCR4 remaining after 6 hours of incubation with SDF, PMA and ionomycin. (F) Cell surface levels of CXCR4 in Jurkat T cells expressing LacZ, Gag-GFP or LTAL Gag-GFP were determined 48 hours post transduction by flow cytometric analysis of cells stained with a biotinylated anti-CXCR4 antibody and Streptavidin-PE. Data shown represents the mean ± SD of surface CXCR4 levels relative to the control, from two independent experiments.