HIV-1 Counteracts GCN2 Inhibitory Effect (E) Wild type (WT) and GCN2 knock out (GCN22/2) MEF were transiently transfected by electroporation with a pcDNA-based plasmid encoding luciferase protein (pcDNA-Luc), as a control, or with an HIV-1 encoding plasmid, pNL4-3.Luc.R-E-. Cells were harvested 24 h after transfection and luciferase activity was measured in cell extracts. Luciferase activity produced in cells from the HIV-1-encoding plasmid was normalized dividing it by the activity produced in cells from pcDNA-Luc plasmid. Results represent the ratio of normalized luciferase activity between wild type MEFs (set as one) and GCN22/2. * P,0.005 vs. WT. Results represent the mean of three to four independent experiments and error bars indicate the standard error (SE). doi:10.1371/journal.pone.0047272.g004 was replaced by the green fluorescent protein (GFP) gene (pNL4- 3.GFP.R-E-), was kindly supplied by Dr. Gustavo del Real (Centro Nacional de Biotecnolog´ıa, Madrid, Spain). Cell culture, virus infection and transfection MEF wild type, GCN2 knock out [39] or with a homozygous knock-in mutation for eIF2alpha (S51A) [40], HeLa, COS-7, BHK-21 and HEK 293T cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) foetal bovine serum (FBS), 2 mM glutamine, antibiotics and non- essential amino acids. MT-2 cells (human T-cell leukemia virus type 1 (HTLV-1)-infected cell line) were maintained in complete RPMI 1640 growth medium (Biochrom AG) supplemented with 5% FBS, 2 mM glutamine, 1% ampicillin, 1% cloxacillin, 0.32% gentamicin. Human Peripheral Blood Mononuclear Cells (PBMC) Figure 5. GCN2 is activated in cells expressing HIV-1 RNA. (A) BHK-C and BHK-A1 cells were transfected with a pcDNA-based plasmid encoding luciferase protein (pcDNA-Luc) or with a mixture of HIV-1 encoding plasmids, pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-. Aliquots of cell lysates containing equal amounts of proteins were analyzed by western blot using specific antisera to detect eIF2alpha phosphorylated on serine 51, phosphorylated or total GCN2, GFP and actin. (B) The same as in (A), but using wild type (WT) and GCN2 knock out (GCN2-/-) MEFs. The membrane was probed with different antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, phosphorylated or total GCN2 and GFP. (C) The same as in (A) and (B), but using HeLa cells. In this case the transfection was done by electroporation. The membrane was probed with different antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, phosphorylated or total GCN2, GFP and actin. Similar results were obtained from duplicate experiments. (D and E) In samples equivalent to those of the experiments described in (A) and (B) where cells were transfected with pNL4-3.Luc.R-E- plasmid, luciferase activity was measured and HIV-1 RNA quantified. Relative values of both are shown as the ratio between GCN2-/- MEFs or BHK-A1 and the corresponding control cells (wild type MEFs or BHK-C), whose value was set as one. Data are expressed as mean 6 SE. * P , 0.05 vs. BHK-C (D) or WT (E). (F) Wild type (WT) and S51A MEFs were transiently transfected by electroporation with a pcDNA-based plasmid encoding luciferase protein (pcDNA-Luc), as a control, or with an HIV-1 encoding plasmid, pNL4-3.Luc.R-E-. Cells were harvested 24 hr after electroporation and luciferase activity was measured in cell extracts. Luciferase activity produced in cells from the HIV-1-encoding plasmid was normalized dividing it by the activity produced in cells from pcDNA-Luc plasmid. Results represent the ratio of normalized luciferase activity between wild type MEFs (set as one) and S51A MEFs. * P , 0.01 vs. WT. doi:10.1371/journal.pone.0047272.g005 PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47272