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

Briefly, Jurkat T cells were transfected with pFRTdual-IRES-HIV and were incubated with different stressinducing agents for the appropriate amount of time, as described above. The medium was replaced with a methionine-free medium supplemented with 10% dialyzed FBS for 30 min, [ 35 S]methionine (150-225 mCi/ml; 1Ci = 37 GBq) (Perkin-Elmer) was next added to the medium, and the cells were incubated for 15 min. Cells were collected by centrifugation, washed two times and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). Radiolabeled proteins were isolated by trichloroacetic acid precipitation on Whatman 3 MM paper. The amount of radioactivity was determined by scintillation counting, and the counts were normalized to protein concentration, which had been determined, using the DC Protein Assay (Bio-Rad).