Figure 2.

Screening of ZFP repressors that inhibit HTLV-I LTR expression. (A) HEK293 cells were transfected with a vector that contains a HTLV-I LTR bidirectionally driving the expression Rluc (anti-sense) and Fluc (sense) luciferase. A mutated Rluc translational start ensures that expression of Rluc only occurs if the 5′ HBZ sequence within the LTR is spliced onto the reporter. A series of HTLV-I ZFP-KRAB repressors (2–10) were transfected with the reporter vector and 48 hrs post-transfection the levels of luciferase were determined. (B) Schematic of the HBZ exogenous vector. (C) HEK293 cells were transfected with a vector containing the HTLV-I 3′-LTR driving the expression of the HBZ-3xFLAG with the ZFP vectors, and 48 hrs post-transfection the levels of HBZ RNA were assessed. Both spliced (HBZsp) and nascent HBZ RNA (HBZusp) was detected. For (A) and (C), error bars represent standard deviation from samples treated in triplicate from two independent experiments. The levels of luciferase or HBZ RNA was made relative to a ZFP-HIV-KRAB control, set a 100%. (D) HEK293 cells were transfected as described in (C) and the HBZ-3xFLAG and ZFPs were detected through their Flag and myc tags, respectively. A Rluc expression vector or untreated cells (mock) were included as ZFP and HBZ detection controls, respectively. Alpha-tubulin was detected as a loading control.