EBV-Encoded Proteins Stabilize HIF1 Alpha Figure 6. Biochemical analysis of EBV-infected and mitogen-activated B-cells, and LCLs. A – Concentration of pyruvate (left panel), lactate (right panel) and lactate dehydrogenase catalytic activity (middle panel) in CD40+IL4-activated (ABC), freshly EBV-infected (EBC) B-cells and LCLs. Notice increased values in LCLs compared with CD40+IL4-activated B-cells, while EBV-infected B-cells showed intermediate values. B – Production of reactive oxygen species in CD40+IL4-activated (ABC), freshly EBV-infected (EBC) B-cells and LCLs. Notice the increased ROS concentration in LCLs, compared with CD40+IL4. Freshly EBV-infected B-cells showed intermediate levels of ROS. doi:10.1371/journal.pone.0042072.g006 It has been shown that tumor cells and rapidly proliferating mitogen-activated T-cells (reviewed in [10,25]) prefer glycolysis even under normoxic conditions; this phenomenon is usually referred to as the Warburg effect. As a result, the cells can produce more NADH (reduced form of the nicotinamide adenine dinucleotide), intermediates for the synthesis of fatty acids, nonessential amino acids and sugars (for nucleotides); this is believed to facilitate cellular protein synthesis. Mitogen- (concanavalin A and IL2) activated rat thymocytes metabolize 90% of their glucose to lactate, whereas nonproliferat- ing thymocytes use only 56% of their glucose supply for lactate production [25]. Thus, only 1.1% of available glucose was oxidized through glycolysis to CO2 by mitogen-activated cells, whereas there was a 27% conversion in resting cells. Consistent with the high rate of anaerobic glycolysis in rapidly proliferating cells, the activities of hexokinase, 6-phosphofructokinase (PFK) and PKLR were increased 10–30-fold relative to their resting state [25]. Many enzymes involved in glycolysis are the products of HIF1A-responsive genes. These include GLUT1, PFK, LDHA1, MCT4 (SLC16A3) and PDK1 (reviewed in [26]). Our findings indicate that under normoxic conditions, aerobic glycolysis is active in LCLs and in freshly EBV-infected B-cells, but not in mitogen-activated B-cells. It has been shown that activation of AKT signaling also results in the induction of aerobic glycolysis [24,26,27], and it was proposed that the AKT pathway could be activated by LMP1 (upon CD40 mimicking, discussed in [28]). Recently, it was shown that the AKT pathway is also activated in EBV-transformed B- cells where it counteracts apoptosis and favors cell survival [29]. LCLs produce at least 15 times more ROS, as a by-product of oxidative metabolism, than mitogen-activated B-cells, and EBV- positive Burkitt’s lymphoma cells produce higher quantities of ROS than EBV-negative Burkitt’s lymphoma cells [30]. LCLs produce much higher levels of ROS than mitogen-activated B-cells without any apparent detrimental effects on the cells. However, LCLs express high levels of LMP1, an EBV-encoded anti- apoptotic protein. EBNA-5 binds to MDM2 and forms trimolec- ular complexes with p53, thereby inhibiting the p53 transcrip- Figure 7. Schematic view on the role of HIF1A in EBV-infected B-cell. A – HIF1A is hydroxylated by the PHDs under normoxic conditions. The hydroxylated HIF1A is recognized by the VHL, E3 ubiquitin ligase, and HIF1A is degraded on proteasomes in activated B-cells. B – Upon EBV infection, EBNA-3 and EBNA-5 bind to PHD2 and PHD1, respectively, and inhibit HIF1A hydroxylation and degradation. The stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT and transactivates genes such as GLUT1, PDK1 and LDHA. This results in conversion of pyruvate to lactate, i.e., aerobic glycolysis. doi:10.1371/journal.pone.0042072.g007 PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e42072