Kinetics and intracellular compartmentalization of HTLV-1 mRNAs; temporal analysis of Tax and Rex protein turnover. (A left) NCN of all HTLV-1 mRNAs in the cytoplasmic and nuclear fractions 24 hours after transfection of HLtat cells with wild-type HTLV-1 molecular clone ACH using Fugene6 (Roche; mean of 3 experiments, standard error bars). NCN values were determined by dividing the absolute copy number of each transcript by the absolute copy number of the GAPDH mRNA. (Right) NCN of all HTLV-1 mRNAs in the cytoplasmic and nuclear fractions of the chronically infected cell line C91PL.¹⁹  (B) Kinetic analysis of the nucleo-cytoplasmic export of the tax/rex, gag, env and HBZ mRNAs expressed from ACH (left) and ACH-Rex-KO (middle) in transfected HLtat cells. RNA was extracted from nuclear and cytoplasmic fractions using the Paris Kit (Ambion). “Export Ratios” were calculated as the ratio between cytoplasmic and total NCN over a time course of 48 hours (harvesting at 0, 16, 24, and 48 hours). The right panel shows Western blot analysis to detect Rex protein (see description in panel E); results verified that the ACH-Rex-KO does not express Rex. (C) Kinetics of Tax and Rex protein expression in HLtat cells from plasmid pBS1–2-3, which contains the viral 5′ and 3′ LTRs and expresses the full-length tax/rex mRNA (including all coding and noncoding regions). Cultures were harvested at 8, 16, 32, and 48 hours after transfection. Cells were fixed in 3.7% formaldehyde-PBS, permeabilized in 0.2% Triton-PBS, blocked with 3% BSA (bovine serum albumin)–PBS and then incubated for 1 hour with mouse anti-Tax monoclonal antibody²³  (1:100, in PBS-1.5% BSA) and rabbit anti-Rex polyclonal antibody²⁴  (1:500, in PBS-1.5% BSA). Cells were next incubated for 1 hour with Alexa 633-conjugated goat anti–mouse and Alexa 488–conjugated chicken anti–rabbit antibodies (Molecular Probes) diluted 1:1000 in PBS-1.5% BSA. Tax and Rex protein expression was analyzed by flow cytometry using a FACSCalibur (Becton Dickinson) equipped with 633-nm Helium-Neon and 488-nm Argon lasers. Alexa 633 and Alexa 488 fluorescent signals were analyzed using the FL4 (661 ± 16 nm) and the FL1 (530 ± 30 nm) detection lines, respectively. Data are represented as equal probability plots. The line graph (right) shows mean and standard error values of Rex/Tax fluorescence intensity (mean fluorescence value X number of positive events) ratios measured in 3 independent experiments. (D) Kinetics of Tax and Rex protein expression in HLtat cells from the infectious HTLV-1 molecular clone ACH. Cultures were harvested at 8, 24, 32, and 48 hours after transfection. Cells were processed and analyzed as described for panel C. Data are represented as equal probability plots. The line graph (right) shows mean and standard error values of Rex/Tax fluorescence intensity (mean fluorescence value X number of positive events) ratios measured in 3 independent experiments. (E) Degradation rates of the Tax and Rex protein expressed from pBS1–2-3 after blocking protein synthesis. HLtat cells transfected with pBS1–2-3 were treated with 10μM cycloheximide 24 hours after transfection and harvested in “disruption buffer” (Paris kit; Ambion) at 3, 8, 24, and 32 hours after cycloheximide treatment. Lysates were subjected to SDS-PAGE and electrotransferred to Hybond-C Extra (GE Healthcare). Blots were incubated with mouse anti-Tax monoclonal antibody (1:500), rabbit anti-Rex polyclonal antibody (1:5000) and mouse anti-tubulin monoclonal antibody (1:2000) in PBS-3% BSA-0.05% Tween followed by a horseradish peroxidase-conjugated anti–mouse or anti–rabbit antibody (Pierce) diluted 1:5000 in 2% milk (Roche)–PBS-0.05% Tween. Blots were developed using chemiluminescence reagents (Supersignal, Pierce) and immunoreactive bands were visualized and quantified using a BioRad ChemiDoc XRS imager. The left panel shows a composite of this Western blot analysis to detect the Tax, Rex and Tubulin signals. Data were normalized by dividing Tax and Rex signals by the tubulin signal and scaled against the value at 3 hours; resulting numbers, which represented the fraction of protein remaining, were plotted in the graph on the right. Protein half-life was estimated by fitting a linear decay model to the data, assuming a constant degradation rate.