FIG. 5.
The NF-κB activation domain of Tax is required for the cooperative transactivation of the HIV-1 LTR. (A) Flag-tagged Tax mutants with mutations in the NF-κB activation domain (M22) and in the transcriptional activation domain (M318). M22 contains amino acid substitutions Ser130 Ala131 for Thr130 Leu131 within the NF-κB activation domain of the Tax protein. M318 has a carboxyl-terminal deletion in the transcriptional activation domain of Tax. Expression of Tax was detected by immunoblotting analysis. The Tax constructs were transiently transfected into 293T cells by using Lipofectamine reagent. The cellular extract prepared from the transfected cells was directly analyzed by immunoblotting with anti-HTLV-I Tax or with anti-Flag. (B) The wild-type Tax or the Tax mutants including M22, M318, and TaxFS (1 μg/each) were cotransfected with the LTR-SEAP reporter plasmid (0.2 μg) and Tat (0.1 μg) into Jurkat T cells. The SEAP assay was performed as described above. The data are representative of at least three independent experiments.