Assessment of MEF-2 binding site(s) within the HTLV-1 LTR. (A) HTLV-1 LTR nucleotide sequence with two putative MEF-2 binding sites shown in blue and red. The HTLV-1 LTR comprises U3 (unique 3′), R (Repeated), and U5 (unique 5′) regions. The U3 region regulates viral gene expression via three 21 bp repeats known as Tax-responsive element - 1 (TRE - 1), which confers Tax-based trans-activation. These repeats contain 3 conserved domains labeled A, B and C. The location for ATF/CREB binding and putative MEF-2 binding are illustrated. (B) EMSA was performed with a probe corresponding to the MEF-2 site in the HTLV-1 LTR using nuclear extracts from Jurkat, MT-2, MT-4 and C8166 cells. (C) EMSA competition assay was performed using nuclear extracts from MT-2 cells, with increasing amounts of unlabeled consensus MEF-2 specific probe (200, 300 and 400 fold molar excess respectively) or a mutated MEF-2 specific probe. Oct-1 was used as a loading control. (D) HTLV-1 LTR luciferase assays in 293 T cells transfected with empty vector (EV) or Flag-Tax using LTR luciferase WT plasmid (LTR Luc WT) or a MEF-2-specific binding mutant (LTR Luc MEF-2 Mut). Luciferase values are presented as “fold induction” relative to the control (EV). Two-tailed unpaired t-test was performed with Prism software. Error bars represent the standard deviation of triplicate samples. The level of significance was defined as: ***p < 0.001. Western blots were performed with anti-Flag and anti-β-actin using whole-cell lysates.