Figure 2. HIF1A binds to EBNA-3, and PHDs 1 and 2 colocalize with EBNA-5 and EBNA-3, respectively. A – GST pulldown assay and Western blotting from lymphoblastoid cell lysates. EBNA-3 was detected in a complex with HIF1A protein. As a positive control, 5% of the crude lysate was loaded. B – Immunoprecipitation and Western blotting from lymphoblastoid cell lysates. HIF1A was detected in a complex with EBNA-3, but not with EBNA-5, on the surface of CNBr beads with immobilized anti-EBNA-3 and anti-EBNA-5 antibodies, respectively. As a positive control, 7% of the crude lysate was loaded. C – Immunostaining of MCF7 cells transfected with GFP-HIF1A (green) and RedDs-EBNA-5 (red). Rabbit anti-PHD1 antibody, followed by biotinylated goat anti-rabbit antibody and AMKA streptavidin (white). EBNA-5 and HIF1A partially colocalize in the nucleus. In doubly transfected cells, the normally cytoplasmic PHD1 is located in the nucleus, colocalized with GFP-HIF1A and RedDS-EBNA-5. D – MCF7 cells transfected with GFP-HIF1A (green) and FLAG-EBNA-3 (white) were stained with rabbit anti-PHD1 antibody, followed by biotinylated goat anti-rabbit antibody and AMKA streptavidin (blue). The mainly cytoplasmic PHD2 redistributed to the nucleus in doubly transfected cells, colocalizing with GFP-HIF1A and FLAG-EBNA-3. doi:10.1371/journal.pone.0042072.g002 Because enhanced ATP synthesis leads to elevated levels of reactive oxygen species (ROS), their levels were assayed in all three groups of cells. Higher levels of ROS were detected in LCLs than in CD40+IL4-activated B-cells, suggesting increased energy production by lymphoblastoid cells (Figure 6B). Freshly EBV- infected cells showed intermediate values. EBV-Encoded Proteins Stabilize HIF1 Alpha Discussion HIF1A is a transcription factor characterized by a basic helix- loop-helix (PAS) domain at the N terminus [14]. It is powerfully induced by hypoxia at both mRNA and protein levels [14]. Under normal conditions, the HIF1A protein level is low because of proteasome-dependent degradation. At high oxygen levels, a few prolines (e.g., residues 402 and 564) are hydroxylated [16,17], leading to the recognition of HIF1A by its E3 ligase, VHL [18], followed by proteasomal degradation. Our findings show that HIF1A is stabilized in EBV-transformed B-cells and, given that mRNA levels are not changed, it seems likely that the stabilization is due to the inhibition of hydroxyl- ation, as we have demonstrated here. We have found, by GST pulldown assay and immunoprecip- itation, that PHDs for HIF1A bind to EBV-encoded proteins involved in growth transformation. Thus, PHD2 binds to EBNA-3 and PHD1 to EBNA-5; this may impair their catalytic activity by steric hindrance. When the cells were subjected to conditions mimicking hypoxia, a small proportion of the available HIF1A was hydroxylated and the levels of both PHD1 and PHD2 increased. Despite binding to EBNA-3, HIF1A was able to transactivate its target genes, including those involved in the glycolytic pathway. PDK1 phosphorylates the mitochondrial pyruvate dehydroge- nase complex responsible for oxidative decarboxylation of pyruvate. The higher level of PDK1 in LCLs compared with mitogen-activated B-cells suggests that the oxidative decarboxyl- ation of pyruvate may be slower in LCLs. The level of GLUT1, a glucose transporter responsible for glucose uptake, is high in thyroid [19] and colorectal cancers [20]. PGK1, a phosphoglyc- erate kinase, is secreted by fibrosarcoma cells at levels several-fold higher than for normal fibroblasts [21]. PKLR catalyzes the conversion of phosphoenolpyruvate and ADP into pyruvate and ATP in the last step of glycolysis. Hexokinase (HK) phosphorylates glucose into glucose-6-phosphate [22], which is the first step of the glycolytic pathway. At high oxygen concentration, glucose is metabolized to CO2 and the cells produce ATP (about 36 molecules per molecule of glucose) and precursors for amino acid synthesis, whereas only minimal amounts of lactate are generated from pyruvate (reviewed in [23]). At low oxygen concentrations, the cells convert most of the pyruvate to lactate and much less ATP is generated (2–4 molecules per 1 molecule of glucose), in a process referred to as anaerobic glycolysis. However, the rate of ATP production is higher and overall metabolism is more efficient during glycolysis (reviewed in [24]). Figure 3. PHDs 1 and 2 bind to EBNA-5 and EBNA-3, respectively. A – Western blotting following the GST pulldown assay from lymphoblastoid cell lysates. PHD2 and a small portion of PHD1 were precipitated on the beads with GST-EBNA-3 from cell lysate. Positive control shows 15% the crude lysate. B – Western blotting following the GST pulldown assay from LCL lysates. PHD1 was precipitated on the support with immobilized GST-EBNA-5 protein from the lymphoblastoid cell lysate. Positive control shows 15% of the crude lysate. doi:10.1371/journal.pone.0042072.g003 PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e42072