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.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an oncogenic protein which has previously been shown to engage the NF-κB, stress-activated MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and extracellular-regulated kinase (ERK)-MAPK pathways. In this study, we demonstrate that LMP1 activates ERK-MAPK in epithelial cells via the canonical Raf-MEK-ERK-MAPK pathway but in a Ras-independent manner. In agreement with the results of a previous study (B. A. Mainou, D. N. Everly, Jr., and N. Raab-Traub, J. Virol. 81:9680-9692, 2007), we show that the ability of LMP1 to activate ERK-MAPK mapped to its CTAR1 domain, the TRAF binding domain previously implicated in PI 3-kinase activation. A role for ERK-MAPK in LMP1-induced epithelial cell motility was identified, as LMP1-expressing cells displayed increased rates of haptotactic migration compared to those of LMP1-negative cells. These data implicate the ERK-MAPK pathway in LMP1-induced effects associated with transformation, suggesting that this pathway may contribute to the oncogenicity of LMP1 through its ability to promote cell motility and to enhance the invasive properties of epithelial cells.

The frequent expression of latent membrane proteins LMP2A and LMP2B in Epstein Barr virus (EBV)-associated tumors suggests that these proteins play a role in EBV-induced epithelial cell growth transformation. Expression of LMP2A and LMP2B had no effect on the morphology of squamous epithelial cells in monolayer culture, but their expression was associated with an increased capacity to spread and migrate on extracellular matrix. Although the mechanisms by which LMP2A and LMP2B promote cell spreading and motility are unclear, the use of selective pharmacological inhibitors has established a role for tyrosine kinases in this phenotype but ruled out contributions of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase/mitogen-activated protein kinase, and protein kinase C. The ability of LMP2B to induce a phenotype that is virtually indistinguishable from that of LMP2A suggests that regions of the LMP2 protein in addition to the cytosolic amino terminus are capable of inducing phenotypic effects in epithelial cells. Thus, rather than serving to modulate the activity of LMP2A, LMP2B may directly engage signaling pathways to influence epithelial cell behavior such as cell adhesion and motility.