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The Epstein–Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.

Background The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-κB. Results In this report we demonstrate that EBNA1 inhibits the canonical NF-κB pathway in carcinoma lines by inhibiting the phosphorylation of IKKα/β. In agreement with this observation we find a reduction in the phosphorylation of IκBα and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-κB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-κB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-κB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied. Conclusions Inhibition of p65 NF-κB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-κB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-κB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.