Virally encoded membrane proteins may also regulate the activity of nuclear receptors. LMP1 can enhance the synthesis of HIF1A (NP_001521) and the expression of HIF1A-responsive genes in an NPC-derived cell line [7], which could be attributed to the enhanced degradation of prolylhydroxylases 1 and 3, mediated by SIAH1 [8]. HIF1A is frequently overexpressed in solid tumors where its levels correlate with tumor progression and poor prognosis in non- small cell lung and colorectal carcinomas, fibrosarcoma and tumors of brain, breast, esophagus, stomach, prostate, ovary, uterus and cervix (reviewed in [9]). HIF1A is one of the subunits of the HIF1 transcription factor. When heterodimerized with aryl hydrocarbon receptor nuclear translocator (ARNT or HIF1B, NP_001659), it activates several genes that mediate oxygen delivery and oxygen utilization under hypoxic conditions. Nor- mally, HIF1A is hydroxylated by prolylhydroxylases 1 and 2 (PHD1, NP_542770; PHD2, NP_071334). The hydroxylated form is recognized by the Von Hippel–Lindau (VHL, NP_000542) protein, an E3 ubiquitin ligase, and degraded by the proteasome machinery. The expression level of HIF1A in lymphoblastoid cells and its function in EBV-induced B-cell transformation have not been previously studied. Given that the malignant or premalignant behavior of EBV-activated B-cells has been a matter of some controversy among pathologists, it is of considerable interest to establish whether the virally activated immunoblasts show a Warburg effect, as with most malignant cells and in contrast to mitogen-activated B-blasts. We here report that HIF1A is stabilized in EBV-transformed lymphoblastoid cells at the protein level. Two virally encoded and growth transformation-associated proteins, EBNA-5 and EBNA-3, bind to prolylhydroxylases 1 and 2, respectively, and interfere with their enzymatic activity. HIF1A is known to transactivate several genes involved in aerobic glycolysis (for a review, see [10]). We also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate. This suggests the activation of the glycolytic pathway, corresponding to the Warburg effect. No such activities were found in mitogen-activated B-cells. Methods Plasmids Cloning of GST2TK-EBNA-5, GST-EBNA-5(2W), RedDS- EBNA-5, GST-EBNA-3, GST-CTEBNA-3 and FLAG-EBNA-3 was described previously. The EBNA-5 constructs contained four W1W2 repeats and the unique C-terminal domain (Y) except GST-EBNA-5(2W), which contained only two W1W2 repeats. GFP-HIF1A, GST-HIF1A and GST-ARNT were constructed as described previously [11,12]. All the constructs were verified by sequencing using a capillary Applied Biosystems sequencing machine (PerkinElmer, Wellesley, MA). Cell lines and transfections The MCF7 breast carcinoma cell line and LCLs (described in [2,6]) were cultured at 37uC in Iscove’s medium containing 10% fetal bovine serum and appropriate antibiotics. Periodic staining with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO) confirmed the absence of mycoplasma. MCF7 cells were grown on coverslips and transfected using Lipofectamine Plus Reagent (Life Technol- ogies, Carlsbad, CA), according to the manufacturer’s protocol. Peripheral B-cells were isolated from buffy coat (Karolinska Hospital, Stockholm) on Lymphoprep gradients. Two subsequent rounds of E-rosetting removed the T-cells. The B95.8 strain of EBV was used for B-cell infection to establish LCLs. For virus- EBV-Encoded Proteins Stabilize HIF1 Alpha unrelated B-cell activation, the cells were exposed to anti-CD40 antibody (Nordic Biosite AB, Ta¨by, Sweden; 1 mg/ml) and interleukin 4 (IL4) (ImmunoTools GmbH, Friesoythe, Germany; 25 ng/ml). All cells were activated by the mitogen, while approximately 40% of cells were infected after 24 h, as shown by immunostaining with anti-EBNA-2. Cells were deposited on glass slides using a Cytospin cytocentrifuge. GST protein production and GST pulldown assay GST fusion proteins were isolated from bacterial cultures (BL21 DE3 strain, Invitrogen, Carlsbad, CA), according to the manu- facturer’s protocol. These proteins were treated with DNAse and RNAse A to reduce nonspecific interference and purified on GST Sephacryl beads (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). A GST pulldown assay was performed as described previously [13]. Western blotting Total cell lysates were prepared using the NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris, pH = 8) with a protease inhibitor cocktail (Roche AB, Stockholm, Sweden) and containing BSA (0.5%(w/v)) as a nonspecific competitor. After SDS–PAGE, proteins were transferred to nitrocellulose and probed with rabbit antibodies against HIF1A, HIF1A-OH (Cell Signaling Technol- ogy, Inc., Billerica, MA), PHD1, PHD2 (Thermo Scientific, Rockford, IL) and EBNA-3 (AsLa Ltd, Riga, Latvia) and with mouse antibodies against HIF1A (Life Technologies), EBNA-5 (DAKO, Glostrup, Denmark) and actin (Sigma-Aldrich). Second- ary antibodies (anti-rabbit and anti-mouse IgG HRP conjugated) were from GE Healthcare Bio-Sciences AB. Immunoprecipitations Antibodies (5 mg of rabbit anti-EBNA-3, mouse anti-EBNA-5 and normal mouse serum (DAKO)) were coupled to CNBr- Sepharose 4 Fast Flow, according to the manufacturer’s protocol (GE Healthcare Bio-Sciences AB). These supports were used with whole cell lysates typically prepared from 107 lymphoblastoid cells. After extensive washing with NP40 lysis buffer and PBS, immune complexes were eluted by heating (94uC) and analyzed on SDS– PAGE. Immunostaining and imaging Immunostaining and digital image capture were performed as described elsewhere. Briefly, cells on coverslips were fixed in a 1:1 mixture of cold methanol and acetone at 220uC. After rehydration in PBS, cells were stained with rabbit anti-PHD1, anti-PHD2 and mouse anti-FLAG (Rockland, Gilbertsville, PA) antibodies. The following secondary antibodies were used: goat anti-rabbit biotinylated, AMKA streptavidin and horse anti-mouse Texas red conjugated (BD Biosciences Pharmingen, San Jose, CA). Images were captured using a DAS microscope, Leitz DM RB, with a dual-mode CCD camera, C4880 (Hamamatsu, Japan). Real-time PCR Total RNA was purified from cells (with and without treatment with NiCl2 (1 mM) for 20 h) using the RNA Purification Kit (Fermentas, Hanover, MD). One microgram of total RNA was used for the cDNA synthesis using a First Strand cDNA Synthesis Kit (Fermentas); for real-time PCR, the total reaction volume was 20 ml and the primer concentration was adjusted to a final concentration of 3 mM. Real-time PCR was performed using SYBR Green Master Mix on a 7900 machine (Applied Biosystems, Foster City, CA). Primers for HIF1A1-responsive genes were the PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e42072