p13 expression decreases viral production and Tax activity. (A) Schematic representation of the HTLV-1 genome that encodes the enzymatic genes gag-pro-pol and env and the regulatory proteins p12, p30, p13, Rex, Tax, and HBZ (arrow indicates antisense direction of the HBZ gene) at the 3′ of the genome. Box under env indicates the second exon that contains the ATG-initiating codons for the envelope p30, Tax, and Rex proteins. The singly spliced orf-II p13 mRNA is illustrated below. (B) Single-letter amino acid code of p13 from HTLV-1_LAF. (C) 293T cells transfected with the HTLV-1 molecular clone pAB (0.5 μg) without and with increasing amounts of p13-HA and the reporter construct HTLV-1 LTR-Luc (0.1 μg). DNA concentrations were equalized using the backbone vector pMH. Viral replication was measured 48 hours after transfection by extracellular p19 Gag ELISA (pg/mL) and intracellular anti-p24 Gag immunoblotting. Lysates were examined for p13 and Tax expression. Tubulin was used as a loading control. Tax-dependent LTR-Luc activity was measured and adjusted for protein concentration. p19 Gag concentration and luciferase activity were set to 100% for pAB alone, and the standard deviation represents an average of 3 independent experiments. (D) Quantitative RT-PCR was performed on cytoplasmic and total RNA isolated from 293T cells transfected with pAB (0.5 μg) and p30, p13, or vector control (pMH) (1.0 μg). RNA levels of cytoplasmic over total for gag (top panel) and tax/rex (bottom panel) are shown. The graphs correspond to gag or tax signals adjusted for gapdh level. Graphs represent values from duplicate experiments. (E) 293T cells were transfected with pcTax (50 ng) without (−) or with (+) increasing amounts of p13-HA and the reporter construct HTLV-1 LTR-Luc (0.1 μg). Luciferase activity is normalized for protein concentration and is the result presented is representative of > 3 independent experiments. Lysates were examined by immunoblotting for expression of p13, Tax, and tubulin.