HBZ RNA inhibits basal and TAX-dependent transactivation of the 5′LTR promoter and decreases sense transcription in HTLV-1–infected C91PL lymphocytes. (A) Schematic representation of wild-type HBZ and mutants (TTG and SM) inserted into pME18Sneo vectors. (B) HEK293 cells were transfected using polyethylenimine (PEI) with 2 µg of vectors encoding TAX (pSGTax) and HBZ (as displayed in panel A). After 48 hours, cell lysates were analyzed by immunoblot using anti-TAX and anti-HBZ antibodies. Tubulin was used as loading control. (C) HEK-LTRLuc cells containing a stably integrated pLTRLuc plasmid were transfected with 1 µg of pME18SNeo (vector), pME18SNeo-HBZ, pME18SNeo-TTG, or pME18SNeo-SM. The firefly luciferase activities (light arbitrary units) were measured at 48 hours posttransfection. (D) HEK-LTRLuc cells were transfected with pSG5, pSGTax, pME18SNeo, pME18SNeo-HBZ, pME18SNeo-SM, and pME18SNeo-TTG vectors (1 µg). (E) At 48 hours, the levels of HBZ RNA were quantified by RT-qPCR using HBZ1 primers (supplemental Table 1) and normalized to those of the HPRT housekeeping gene. (F) In parallel, TAX expression was determined by immunoblot as described in panel B. Numbers indicate the mean quantification of band luminescence intensities of TAX relative to tubulin resulting from 3 independent immunoblots. (G-I) HTLV-1 immortalized lymphocytes C91PL (not transduced [NT]) were stably transduced with pUCOE-SFFV-EGFP, pUCOE-SFFV-HBZ, pUCOE-SFFV-TTG, and pUCOE-SFFV-SM lentiviruses expressing mock (enhanced green fluorescent protein [EGFP]), HBZ TTG or SM, respectively. The leukemic HTLV-1–infected ATL2 cell line was analyzed in parallel as reference for physiological HBZ RNA levels. HBZ (G), TAX (H), and GAG (I) RNA levels were quantified by RT-qPCR and normalized to HPRT. All data are presented as mean plus or minus standard error of at least 3 independent experiments. Statistical P values were calculated according to the Tukey multiple comparison tests.