Epstein-Barr virus-encoded EBNA1 inhibits the canonical NF-κB pathway in carcinoma cells by inhibiting IKK phosphorylation

Robert Valentine,Christopher W Dawson,Thomas J Owen,Kathryn L Date,Khilan M Shah,Chunfang Hu,John R Arrand,Sonia P Maia,John D O'Neil,Lawrence S Young,Jianyong Shao

doi:10.1186/1476-4598-9-1

Abstract

BackgroundThe 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.ResultsIn 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.ConclusionsInhibition 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.

Highlights

The Epstein-Barr virus (EBV)-encoded Epstein-Barr nuclear antigen-1 (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
Cell lines and tissue culture Ad/AH, Hone1, AGS and derivatives stably expressing EBNA1 at levels comparable to those found in EBV infection, Ad/AH cells stably infected with a recombinant EBV and C666-1 were cultured as previously described [13,14]
EBNA1 represses p65 NF-B activity in carcinoma cells To assess whether EBNA1 influenced NF-B activity we initially performed luciferase reporter assays in a range of carcinoma cell lines using a synthetic NF-B reporter and found that NF-B activity in Ad/AH, AGS and Hone1 cells stably expressing levels of EBNA1 comparable to those found in EBV infection was inhibited by 8, 5 and 2.6 fold, respectively (Fig. 1)

Summary

Introduction

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. In addition to EBNA1’s functions that depend on its binding to viral DNA, EBNA1 can interact with host cell proteins, including the ubiquitin-specific protease USP7 which has been implicated in the destabilisation of p53 by binding with a higher affinity to the same region of USP7 as do p53 and MDM2 This suggests that EBNA1 can protect against either UV- or p53-induced apoptosis [4]. In support of a role for EBNA1 in carcinogenesis we and others have demonstrated that EBNA1’s transcription factor-like functions are not confined to the regulation of viral genes and extend to the regulation of host cell gene expression This has been demonstrated in the context of B-cells where EBNA1 has been shown to induce the expression of CD25, RAG1, RAG2 and CCL20 [10,11,12] whilst in epithelial cells we have established that expression of EBNA1 results in the differential regulation of cellular genes involved in translation, transcription and cell signalling [13,14]. Potential EBNA1 binding sites have been found in the promoters of numerous other cellular genes [15]

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Conclusion