The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element

Gendron, Karine; Ferbeyre, Gerardo; Heveker, Nikolaus; Brakier-Gingras, Léa

Jurkat T cells (CD4+ T cells) were maintained in RPMI 1640 medium (Gibco) supplemented with 10% (v/v) FBS (Wisent). All transfections were made by electroporation, using the Neon TM transfection system (Invitrogen) according to the manufacturer's instructions. The conditions for electroporation were one pulse of 1150 V for 40 ms. Transfections were performed in 24-well plates containing 1.5 Â 10 5 Jurkat T cells in 1 ml of complete medium without antibiotics. Briefly, 1 mg of DNA was mixed with 1.5 Â 10 5 Jurkat T cells in 10 ml of R buffer supplied by the manufacturer. For assays with shRNA, co-transfections with shRNA-encoding plasmids were performed in 6-well plates containing 2.0 Â 10 6 Jurkat T cells in 5 ml of complete medium without antibiotics. Two micrograms of dual-luciferase reporter were mixed with 2 mg of the plasmid encoding either control shRNA (pBS-U6-ApaI) or shRNA targeting Rluc (pBS-U6-RLi), a gift from Ivan Shatsky. Each mix was added to 2.0 Â 10 6 Jurkat T cells in 100 ml of R buffer. Transfections for treatments with chemical agents were performed in 6-well plates containing 2 ml of complete medium without antibiotics. Four mg of dual-luciferase reporter were mixed with 1.0 Â 10 6 Jurkat T cells in 10 ml of R buffer. Twenty-four hours post-transfection, transfected cells were pooled and splitted in 24-well plates (500 ml per well) and the chemical agents (H 2 O 2 at final concentrations of 5 and 10 mM, TBHQ at final concentrations of 150 and 300 nM, thapsigargin at a final concentration of 450 nM and DFX at final concentrations of 250 and 500 mM; control: DMSO 0.25%) were added in a final volume of 1 ml per well for 4-8 h, as indicated in the legend to Figure 3 .