EBV-Encoded Proteins Stabilize HIF1 Alpha Figure 1. Expression level of HIF1A in EBV-infected and mitogen-activated B-cells, and in LCLs. A – Western blotting of HIF1A and its hydroxylated form in LCLs (left panel). The membrane was probed with mouse antibody against HIF1A and the rabbit serum against the hydroxylated form of HIF1A (HIF1A-OH). Notice the absence of HIF1A-OH in LCLs (first lane) and the lack of effect of proteasome inhibition. In contrast, high levels of hydroxylated HIF1A were detected in control MCF7 cells upon proteasome inhibition (right panel). B – Western blotting of whole cell lysates of CD40+IL4-activated B-cells, freshly EBV-infected B-cells and LCLs. Cells were treated with 1 mM NiCl2, mimicking hypoxia-like conditions. Notice that HIF1A, PHD1 and PHD2 levels in LCLs did not change upon treatment with NiCl2, in contrast to freshly infected cells. C – HIF1A expression in CD40+IL4-activated (ABC), freshly EBV-infected (EBC) B-cells and LCLs (LCL) measured by Q-PCR. doi:10.1371/journal.pone.0042072.g001 Prolylhydroxylase 1 and 2 bind to EBNA-5 and EBNA-3, respectively. To verify that EBNA-3 may form a complex with PHD2 and EBNA-5 with PHD1, a GST pulldown assay was performed using lymphoblastoid cell lysates. GST tagged with intact EBNA-3, the C-terminal region of EBNA-3, EBNA-5 containing either 2 or 4 W1W2 repeats, and GST alone on the beads were used in a pulldown assay. Substantial amounts of PHD2 were precipitated with EBNA-3, whereas PHD1 formed complexes with EBNA-5 (Figure 3A and B). These results indicate that HIF1A is stabilized in LCLs because of a lack of hydroxylation, which may in turn result from an inhibition of the catalytic activity of PHDs due to binding of cognate EBNAs. Nuclear HIF1A pool is enriched in LCLs. To test directly whether the level of HIF1A is increased in the nucleus of LCLs, we separated cytoplasmic and nuclear fractions from LCLs and CD40+IL4-activated cells. Western blotting with anti-HIF1A antibody showed an increase in nuclear HIF1A in LCLs (Figure 4A), and similar conclusions were drawn from immuno- staining experiments (Figure 4B). Conversely, the HIF1A signal (green) was observed mainly in the cytoplasm of mitogen-activated B-cells, while in LCL it showed both a cytoplasmic and a nuclear distribution. HIF1A is constitutively transcriptionally active in LCLs. To test whether the transactivating ability of HIF1A was inhibited in the LCLs, Q-PCR was performed for HIF1A- responsive genes (Figure 5). LCLs were compared with both CD40+IL4-activated and freshly EBV-infected B-cells. Given that EBV-infected immunoblasts are not normally exposed to hypoxia either in vitro or in their natural niche in vivo, and given the involvement of HIF1A in glycolysis, we examined the expression of several genes of the glycolytic pathway (Figure 5). GLUT1 (glucose transporter), LDHA (lactate dehydrogenase), PDK1 (pyruvate dehydrogenase kinase), PGK1 (phosphoglycerate kinase), PFKL (phosphofructokinase), SLC16A3 (MCT4, monocar- boxylate transporter), HK1 (hexokinase) and PKLR (pyruvate kinase) were expressed at levels 5–150-fold higher in freshly EBV-infected cells and in LCLs than in CD40+IL4-activated B- cells. These genes encode essential enzymes involved in glycolysis: GLUT1 controls glucose transport; PDK1 catalyzes oxidative decarboxylation of pyruvate; and LDHA controls a step in anaerobic glycolysis by catalyzing the conversion of L-lactate and NAD into pyruvate and NADH, respectively. LCLs show a ‘‘Warburg effect’’. To test whether LCLs show a ‘‘Warburg effect’’ (i.e., a preference for aerobic glycolysis, similar to that seen in solid tumors), we measured lactate and pyruvate levels and LDHA enzymatic activity in cultured cells. CD40+IL4-activated cells were compared with freshly EBV- infected B-cells and LCLs cultured under the same conditions. The concentrations of lactate and pyruvate were 5- and 3-fold higher in the LCLs than in mitogen-activated B-cells. The catalytic activity of LDHA was at least 50-fold higher in LCLs, compared with CD40+IL4-activated B-cells. EBV-infected B-cells showed intermediate values for lactate, pyruvate and lactate dehydroge- nase (Figure 6A). PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e42072