HIV-1 Counteracts GCN2 Inhibitory Effect Figure 4. Absence of GCN2 favor HIV-1 protein expression. (A, B) HeLa cells were co-transfected with plasmids encoding wild type GCN2 or the inactive mutant GCN2-K618R and also with a mixture of HIV-1 encoding plasmids pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-. Cells were harvested 24 h after transfection. Aliquots of cell lysates containing equal amounts of proteins were analyzed by western blot using specific antisera to detect GCN2, eIF2alpha and GFP (A). Luciferase activity was measured, and results are shown as the ratio between the activity in cells over-expressing GCN2-K618R mutant and in cells over-expressing wild type GCN2. * P,0.05 vs. WT (B). (C, D) Normal HeLa cells (no siRNA) and HeLa cells stably expressing a control siRNA (siRNA 3) or a specific siRNA for GCN2 knock down (siRNA 4) were transiently transfected with a mixture of HIV-1 encoding plasmids, pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-. Cells were harvested 24 h after transfection. Aliquots of cell lysates containing equal amounts of proteins were analyzed by western blot using specific antisera to detect GCN2, eIF2alpha, GFP and HIV-1-p24 (C). Luciferase activity was also measured, and results are shown as the ratio between the activity in cells expressing siRNA 3 or siRNA 4 and in normal cells (no siRNA). * P,0.01 vs. no RNAi and RNAi 3 (D). PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47272