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. 2012;7(7):e42072.
doi: 10.1371/journal.pone.0042072. Epub 2012 Jul 27.

Epstein-Barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent with the Warburg effect

Suhas Darekar  1 , Konstantinos Georgiou, Mariya Yurchenko, Surya Pavan Yenamandra, Georgia Chachami, George Simos, George Klein, Elena Kashuba
Affiliations

Epstein-Barr virus immortalization of human B-cells leads to stabilization of hypoxia-induced factor 1 alpha, congruent with the Warburg effect

Suhas Darekar et al. PLoS One. 2012.

Abstract

Background: Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.

Methods and findings: Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.

Conclusions: Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
Figure 4. HIF1A is localized to the nucleus and cytoplasm in LCLs, in contrast to mitogen-activated B-cells.
A – Western blotting of cellular subfractions. The membrane was probed with mouse anti-HIF1A and anti-actin antibodies (left panel). Nuclear HIF1A was detected in the lysates of LCL; however, no nuclear signal for HIF1A was detected in mitogen-activated cells. The HIF1A/actin ratio for all the probes was calculated (right panel). B – Immunostaining of LCLs and mitogen-activated cells with mouse anti-HIF1A antibody (green signal). Nuclear DNA is stained in blue. Notice the presence of nuclear HIF1A signal in LCL (panels a and b) and its absence in the mitogen-activated cells (c and d).
Figure 5
Figure 5. Expression of HIF1A-responsive genes in EBV-infected and mitogen-activated B-cells and LCLs.
Expression of HIF1A-responsive genes, assessed by Q-PCR. CD40+IL4-activated (ABC) and freshly EBV-infected (EBC) B-cells were compared with LCLs. Untreated and cells treated with NiCl2 were compared. Notice that many genes are upregulated in LCL and freshly EBV-infected cells, compared with CD40+IL4-activated B-cells.
Figure 6
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.
Figure 7
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.
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This work was supported by the Swedish Cancer Society, by matching grants from the Concern Foundation (Los Angeles) and the Cancer Research Institute (New York), by the Swedish Institute, and by Karolinska Institutet. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.