FIG. 4.

TORC coactivators are required for optimal activation of the HTLV-1 LTR by Tax. (A to C) Inhibition of TORC-dependent transcriptional activity by shRNAs. Jurkat cells were transfected with reporter plasmid pLTR-Luc (8 μg), a fixed amount (2 μg) of expression plasmids for TORC1 (A), TORC2 (B), and TORC3 (C), and escalating amounts (2 to 10 μg) of plasmids expressing shRNA against TORC1, TORC2, or TORC3 (shTORC). (D) Depletion of TORCs by shRNAs. HeLa cells were transfected with expression plasmids for GFP-tagged TORC1 (lanes 1 to 3), TORC2 (lanes 4 to 6) and TORC3 (lanes 7 to 9) as well as empty shRNA expression vector (vec; lanes 1, 4 and 7), expression vector for shRNA targeting luciferase (shLuc; lanes 2, 5, and 8), and expression plasmid for shRNA against TORC1, TORC2, and TORC3 (shTORC1/2/3; lanes 3, 6, and 9), respectively. Expression of GFP-TORC proteins (upper panels) and antitubulin (tub; lower panels) was analyzed by Western blotting. (E) TORCs are required for Tax activation in T lymphocytes. Jurkat lymphocytes were electroporated with reporter plasmid pLTR-Luc (8 μg), Tax expression plasmid pIEX (1 μg), and increasing amounts (2 to 10 μg) of a mixture of plasmids expressing shRNAs targeting TORC1, TORC2, and TORC3 (shTORC1 to -3). The shRNA against GFP (shGFP) was also included as a control. Cells were harvested 48 h after electroporation, and relative luciferase activities (RLA) were measured. The results represent three independent experiments and the error bars indicate standard deviations. −, absence of indicated protein; +, presence of indicated protein.