Figure 4

Non-productive RGH infection inversely correlates with both cellular activation and NFκB activity. A: RGH infected Jurkat cells (four days post-infection) were assayed for cellular activation by staining with anti-CD69 antibodies and analysis by flow cytometry. Uninfected cells were treated with either DMSO or PMA/Ionomycin for 24 hours prior to analysis. Error bars represent standard deviations of triplicate experiments. * p < 0.05, *** p < 0.001 (Student’s T test). B: Four days post-infection, RGH infected Jurkat cells were stained for NFκB p50 (left panel) and p65-S529-phospho (right panel) subunits and analyzed by flow cytometry. Uninfected cells were treated with DMSO or PMA/Ionomycin for 30 min prior to analysis. Error bars represent standard deviations of triplicate experiments. ‘ns’ non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s T test). C: Schematic representation of Red-Blue-HIV-1 (RBH) and NFκB-eGFP reporter cell lines. RBH contains tagBFP in place of eGFP but is otherwise isogenic. Jurkat NFκB reporter cell lines contain a stably integrated eGFP marker driven by an NFκB responsive promoter (4x tandem NFκB cis-elements ‘GGGACTTTCC’ upstream of a CMV minimal promoter). D: Jurkat cells were infected with comparable amounts of RGH and RBH viral stocks. Cells were analyzed by flow cytometry four days post-infection. Plots shown are representative of multiple independent infection experiments. E: Jurkat NFκB reporter clone 1 was treated with DMSO, TNFα, or PMA/Ionomycin for 24 hours prior to analysis by flow cytometry for eGFP mean fluorescence intensity (MFI). Error bars represent one standard error of the mean. *** p < 0.001 (Student’s T test). F: Jurkat NFκB reporter clone 1 was infected with RBH and analyzed by flow cytometry at four days post-infection. Cells were gated into their constituent infected populations and then analyzed for eGFP MFI. Error bars represent one standard error of the mean. *** p < 0.001 (Student’s T test).