Mutagenesis analysis of Nef residues involved in NFAT activation. (A) Steady-state expression levels of different Nef proteins examined by Western blotting (W.B.). Results are representative of three independent transfection experiments. (B) Jurkat T cells were transfected with an NFAT (ARRE2)-luciferase reporter together with an empty control vector (control), a wild-type (WT) allele, or different mutant HIV-1 Nef alleles as indicated. Twenty hours later one of the control vector-transfected cultures was left untreated while the other, together with the rest of the cultures, was treated with 100 ng of PMA/ml for 5 h. Luciferase activity of the PMA-treated, wild-type Nef-transfected cells was set to 100%, and the activities of other samples are shown relative to this. The luciferase data represent mean values from at least four independent transfection experiments. Variation between these experiments is indicated by standard error bars.

Synergistic activation of NFAT is a conserved function of Nef. Jurkat T cells were transfected with an NFAT-dependent ARRE2 luciferase reporter plasmid together with an empty control vector (control) or with vectors for different HIV-1 or SIV Nef proteins as indicated. Twenty hours after transfection one of the control vector-transfected cultures was left untreated while the other, together with the rest of the cultures, was treated with 100 ng of PMA/ml for 5 h. The mean induction (fold increase) in relative luciferase activity in each case is shown and represents data from four or more independent transfection experiments. Variation between different experiments is indicated by standard error bars.

Overexpression of a constitutively active form of PKC-θ-A148E efficiently synergizes with Nef to activate NFAT-dependent gene expression. (A) Jurkat T cells were transfected with an NFAT (ARRE2)-luciferase reporter with (solid bars) or without (open bars) a PKC-θ-A148E (θDA) expression plasmid. When indicated, the cells were also cotransfected with Nef. An empty control plasmid was used to normalize the total amount of DNA used per transfection. Twenty hours later some of the cultures that were not transfected with Nef were treated with 1 μg of ionomycin (iono)/ml for 5 h while the others (untreated) as well as the Nef-transfected cultures were left untreated. These data are plotted as fold induction relative to luciferase activity measured from untreated cells transfected with the control plasmid. This experiment was repeated four times with similar results. (B) Jurkat T cells were transfected with an NFAT (ARRE2)-luciferase reporter and Nef together with constitutively active PKC isoforms PKC-α-A25E (αDA), PKC-δ-A147E (δDA), and PKC-θ-A148E (θDA) or an empty vector (control). Twenty hours later one of the control transfected samples was treated with 100 ng of PMA/ml for 5 h (PMA). These data are plotted as in panel A and are from four independent transfection experiments. (C) 293T fibroblast cells were transfected with a serum response element (SRE)-luciferase reporter construct together with constitutively active PKC isoforms and treated as explained above. These data represent results from two independent transfection experiments.