Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect Suhas Darekar1, Konstantinos Georgiou 1 , Mariya Yurchenko2, Surya Pavan Yenamandra1¤a, Georgia Chachami3,4¤b, George Simos3,4, George Klein1., Elena Kashuba1,2*. 1 Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden, 2 R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU, Kyiv, Ukraine, 3 School of Medicine, University of Thessaly, Larissa, Greece, 4 Institute of Biomedical Research and Technology (BIOMED), Larissa, Greece 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. Citation: Darekar S, Georgiou K, Yurchenko M, Yenamandra SP, Chachami G, et al. (2012) Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect. PLoS ONE 7(7): e42072. doi:10.1371/journal.pone.0042072 Editor: Sai Wah Tsao, The University of Hong Kong, China Received February 2, 2012; Accepted July 2, 2012; Published July 27, 2012 Copyright: 2012 Darekar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: 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. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Elena.Kashuba@ki.se . These authors contributed equally to this work. ¤a Current address: Bioinformatics Institute, Matrix, Singapore ¤b Current address: Zentrum f u¨r Molekulare Biologie, Universita¨t Heidelberg (ZMBH), Heidelberg, Germany Introduction Epstein-Barr virus (EBV) is a ubiquitous human gamma herpesvirus that causes lymphoproliferative disease in immuno- suppressed patients and is associated with Burkitt’s lymphoma, Hodgkin’s lymphoma, other B- and T-cell lymphomas, nasopha- ryngeal carcinoma (NPC) and some gastric carcinomas [1]. EBV can infect B-cells in vitro and convert them into immortalized lymphoblastoid cell lines (LCLs). LCLs express six nuclear proteins (EBNA-1–6) and three latent membrane proteins (LMP-1, -2A and -2B). This growth transformation program is referred to as Latency III. Six of the proteins, EBNA-1, -2, -3, -5, -6 and LMP-1, are required for efficient B-cell transformation [1]. Expression of the six transformation-associated proteins leads to extensive changes in cellular gene expression and signaling. The EBNAs interact with a variety of cellular proteins and play different roles in the viral strategy. EBNA-1 is essential for the maintenance of viral episomes and regulates viral promoter usage; EBNA-2 activates the viral LMP-1 promoter and a number of cellular genes, such as CD23, C-MYC, cyclin D1 and HES. EBNA-5 enhances EBNA-2-dependent transcription and inactivates p53 by forming a trimolecular complex with MDM2 and p53 [2]. EBNA- 6 is involved in chromatin remodeling and the regulation of the RB pathway (for a review, see [3]). The EBNA-3 family proteins (EBNA-3, -4 and -6) regulate the transcription of viral and cellular genes and are involved in the control of G2/M transition (reviewed in [3]). EBNA-3 also participates in the regulation of transactivation by several nuclear receptors. It binds to aryl hydrocarbon receptor (AHR) (NP_001612) and its regulatory protein XAP-2 (NP_003968) [4] and thereby enhances the constitutive and ligand- (e.g., TCDD) activated transcription of AHR-responsive genes [5]. Moreover, EBNA-3 blocks the transcription of VDR-responsive genes, and thereby protects LCLs against the growth arrest and/or apoptosis induced by the interaction of vitamin D3 with its receptor (VDR, NP_000367) [6]. PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e42072