USF-1 binds HIV core promoter but is dispensable for Tat trans -activation. (A) Mutations in TASHET used in EMSA and transient transfections shown in B, C and D: the name of the mutation is indicated at the left of the sequence. The mutated nucleotides are indicated in white. The TATA box is delimitated by a purple box and the E-box by a green box: the lighter green region indicates nucleotides not forming part of the core E-box consensus sequence. (B) EMSAs were performed as in Figure 2. The different radiolabelled HIV promoter mutations used as a probe are indicated at the bottom of the figure. Supershift experiments were performed with the indicated antibodies. PICHs are indicated by an arrow at the left of each panel. An X in red indicates PICHs whose formation is severely impaired by the mutations. (C) HeLa cells have been co-transfected with a plasmid expressing the Renilla luciferase under the control of HIV wt or mutated promoter with or without a Tat expression plasmid as indicated under the X-axis. Luciferase activity was measured 48 h after transfection in cell extracts. Basal (−) and Tat-induced (+) activities are displayed. Mutation in the TATA box is shown in purple, mutations in the E-box are indicated in green. (D) As in C except that results are expressed as Tat trans-activation, obtained by the ratio of RLU in presence of Tat versus without Tat. Values are expressed as a percentage of wt HIV promoter luciferase activity (100%).