Figure 3.

Anti-HTLV-I ZFP repressors inhibit the LTRs from multiple HTLV-I genotypes. (A) HEK293 were transfected with a vector containing the HTLV-1 LTR bi-directional reporter to measure Fluc (sense) or the HBZ(spliced)-Rluc (anti-sense) activity with the modified ZFP5 vectors, and at 48 h post-transfection the levels of luciferase activity were assessed. The modified vectors were generated by fusing the ZFP5 to a KRAB(ZIM3), KRAB-meCP2 or PAM. A ZFP5 without a KRAB domain was also included (–). The levels of HBZ (B) protein or (C) RNA were determined after transfecting HEK293 cells with an LTR-HBZ and the modified ZFP5 vectors. (D) A schematic of the HTLV-I LTR bidirectional vector with the LTR upstream of the HBZ translation start replaced with sequences from different HTLV-I genotypes (a-g). The country of viral origin, accession numbers, genotypes and ZFP5 target site sequences are indicated. Mismatches are highlighted in red. (E) HEK293 cells were transfected with an LTR(a-g) spliced reporter vector with the ZFP5-KRAB and ZFP5-KRAB-meCP2 vectors, and 48 h post-transfection the levels of Rluc and Fluc luciferase was determined. For (A), (C) and (E), the ZFP5 vectors were made relative to a control ZFP-HIV-KRAB, which was set a 100%. Error bars represent standard deviation from samples treated in triplicate. For (B), the HBZ and ZFPs were detected through a FLAG tag and myc tag, respectively. Untreated cells (mock) were included as ZFP and HBZ detection controls and α-tubulin as a loading control.