p13 modulates virus expression in HTLV-1–infected cells. (A) 293T cells were transfected with HTLV-1 molecular clones pAB (gray bars) or p13KO (black bars). Culture supernatants were harvested 48 and 72 hours after transfection and extracellular p19 Gag levels measured by ELISA assay. Western blot analysis for intracellular p24 Gag, Tax, and actin was performed on whole-cell lysates from 72-hour cultures. The values given are an average of 4 independent experiments. (B) Luciferase activity for the reporter construct LTR-Luc (0.5 μg or 1.0 μg) was measured for 293T cells transfected with HTLV-1 molecular clones pAB (gray bars) or p13KO (black bars). Values are an average of 2 independent experiments and are adjusted for transfection efficiency using pRLTK-Luc. (C) MT-2 cells were transfected with the reporter construct HTLV-1 LTR-Luc and without (−) and with increasing amounts of p13-HA. Lysates were immunoblotted for the expression of p24 Gag, p13-HA, and tubulin. Luciferase activity was measured 48 hours after transfection and normalized for protein concentration. Luciferase activity was set at 100% for cells transfected in the absence of p13-HA. SD is given for the average of 3 independent experiments. (D) MT-2 cells were transfected with p13-HA and, 48 hours after transfection, cells were added to fibronectin-precoated coverslips, fixed, and stained with anti-HA, anti-Tax, or anti-SC35 antibodies for analysis by confocal microscopy. DAPI staining identifies the cell nucleus. (E) Schematic representation of the predicted interplay between Tax and p13. Tax binds to the viral LTR in the nucleus through interaction with CREB. Tax activates transcription through the recruitment of basal transcription machinery and coactivators such as p300/CBP. The p13 protein expressed alone resides in the mitochondrial membrane, affects the electron transport chain (ETC), the membrane potential (Δψ), and the production of reactive oxygen species (ROS). In the presence of Tax, however, p13 is stabilized, becomes ubiquitinated, and part of it interacts with Tax, colocalizes with Tax to nuclear speckles, and reduces viral expression.⁶⁰