Schematic view on the role of HIF1A in EBV-infected B-cell.

<p><b>A</b> – 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>B –</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 <i>GLUT1</i>, <i>PDK1</i> and <i>LDHA</i>. This results in conversion of pyruvate to lactate, i.e., aerobic glycolysis.</p

Expression of HIF1A-responsive genes in EBV-infected and mitogen-activated B-cells and LCLs.

<p>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.</p

Expression level of HIF1A in EBV-infected and mitogen-activated B-cells, and in LCLs.

<p><b>A –</b> 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>B –</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. <b>C – </b><i>HIF1A</i> expression in CD40+IL4-activated (ABC), freshly EBV-infected (EBC) B-cells and LCLs (LCL) measured by Q-PCR.</p

HIF1A is localized to the nucleus and cytoplasm in LCLs, in contrast to mitogen-activated B-cells.

<p><b>A –</b> 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>B –</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 <b>a</b> and <b>b</b>) and its absence in the mitogen-activated cells (<b>c</b> and <b>d</b>).</p

PHDs 1 and 2 bind to EBNA-5 and EBNA-3, respectively.

<p><b>A –</b> 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>B –</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.</p

Biochemical analysis of EBV-infected and mitogen-activated B-cells, and LCLs.

<p><b>A –</b> 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>B –</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.</p

Frequencies of HIF-1α polymorphisms between ON patients and controls.

*<p>Frequencies of rare alleles.</p>**<p>P values of deviation from HWE, in patients and controls.</p

Analysis of HIF-1α polymorhisms using RFLP.

<p>(A) Analysis of HIF-1α gene C1772T polymorphism with HphI enzyme, shown on 2% agarose electrophoresis. CT heterozygous genotype yielded three bands 467, 251 and 216 bp; C allele wt yielded two bands (251 and 216 bp). T allele remained uncut and yielded one fragment at 467 bp. Molecular weight standards are shown on the right. (B) Chromatograms of DNA sequence analysis of HIF-1α exon12 fragment showing the corresponding C, CT, T allelic variations at position 1772. (C) Analysis of HIF-1α gene G1790A polymorphism with AciI enzyme, shown on 2% agarose electrophoresis. G allele wt yielded two bands (269 and 198 bp). A allele would remained uncut and yielded one fragment at 467 bp, as represented. Molecular weight standards are shown on the right. (D) Analysis of HIF-1α gene C111A polymorphism with BglII enzyme, shown on 2% agarose electrophoresis. C allele wt yielded two bands (143 and 44 bp). A allele would remained uncut and yielded one fragment at 187 bp. Molecular weight standards are shown on the right.</p

P582S and A588T mutations do not affect HIF-1α transcriptional activity, localization and expression.

<p>HIF-1α transcriptional activity was determined 24 h after co-transfection of Saos-2 (A) and HEK293 cells (B) with plasmids expressing the indicated proteins together with the pGL3–5HRE-VEGF reporter plasmid. Values (as relative luminescence units) were determined as a ratio of firefly luciferase activity over Renilla luciferase activity and represent the mean of four different experiments performed in triplicate (+/- S.E.). Statistical differences were assessed using unpaired-t-test. P values>0.05 were considered as statistically non-significant (ns). Saos-2 (C) and HEK293 cells (D) where transfected with plasmids expressing the indicated proteins and their localization was observed after 24 h, with immunofluoresence, using an anti-FLAG antibody. Protein levels of FLAG-HIF-1α wt and mutants were determined in Saos-2 (E) and HEK293 (F) cells after 24-48 h post-transfection, in Western blot using a monoclonal anti-HIF-1α antibody and anti-tubulin as loading control. Densitometric analysis of the bands was performed with the public domain software for image analysis ‘ImageJ’. FLAG-HIF-1α quantities were normalized against corresponding tubulin and expressed as fold increase against wt FLAG-HIF-1α (100%).</p

HIF-1α SNP genotype and allele frequencies between ON patients and control subjects.

*<p>Fisher chi-square analysis, P values <0.05 are considered as statistically significant.</p>**<p>OR, Odds ratio; CI, confidence interval.</p><p>Calculations were performed for genotypes: CC vs CT+TT, GG vs GA+AA.</p><p>nc: not calculated.</p