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

Transfected Jurkat T cells were harvested 24 h posttransfection, centrifuged at 200 g for 5 min, washed with PBS and lysed in 100 ml of Cell Passive Lysis Buffer (Promega). Cell lysates were frozen at À80 C until being used. Prior to luciferase assays, cell lysates were thawed and centrifuged 2 min at 1300 g to remove cell debris. The Fluc and the Rluc activities were assayed using a Dual-Luciferase Reporter Assay System kit (Promega). The IRES activity was monitored as a Fluc/Rluc ratio where the Fluc activity is the readout for IRES-dependent translation while Rluc expression depends upon cap-dependent translation. Fluc and Rluc activities were measured as relative light units with a Triathler TM multilabel tester (Hidex Oy). One-way ANOVA with Bonferroni's multiple comparison test was performed using GraphPad Prism version 5.00 for Mac OS X.