VNP-Nefs cannot prevent activation-induced apoptosis of infected cells. (A, B) Expression of (A) CD69 and (B) CD25 at one or two days, respectively, after the second PHA stimulation in PBMCs transduced with HIV-1 IRES/eGFP constructs expressing the indicated groups of Nefs. (C, D) Correlations between expression of CD25 and (C) CD69 or (D) Nef-mediated CD28 down-modulation. (E) Quantitative assessment of levels of apoptosis two days post-stimulation in HIV-1-infected PBMCs. (F-H) Correlation between apoptosis and expression of (F) CD69 and (G) CD25 or (H) CD28 down-modulation by the respective Nefs. (I) PHA-induced NF-AT-dependent luciferase activity in HIV-1 infected Jurkat cells stably transfected with an NF-AT-dependent reporter gene . (J) Correlation between CD69 expression and NF-AT-dependent luciferase expression. (K) Nef-mediated activation of NF-κB in 293Ts co-transfected with a NF-κB-dependent firefly luciferase construct, a pTAL promoter gaussia luciferase construct and an expression vector expressing the various Nef proteins in the presence of TNFα. The assay was performed as described elsewhere . Panels A, B, E, I and K give average values (±SD) relative to those obtained with the nef-defective control HIV-1 construct and were derived from three independent experiments. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.