The role of latent membrane protein 2 in the pathogenesis of Epstein-Barr virus associated malignancy

Abstract

Epstein-Barr virus (EBV) establishes a lifelong latent infection in host B cells and is associated with the development of various lymphoid and epithelial malignancies. Approximately half of Hodgkin's lymphomas (HL) are EBV positive where LMP2 is highly expressed. However, the contribution of LMP2 to the pathogenesis of HL remains largely unknown. In this study, HL cell lines stably expressing LMP2A and LMP2B HL were generated. The impact of global gene expression in KMH2 cells expressing LMP2A and LMP2B was performed using Gene expression microarrays; this identified a number of potentially important transcriptional targets of LMP2A and LMP2B which may be of relevance to the development of HL, but also highlighted important difference in the effect of these proteins on cellular gene expression that were dependant on their expression levels. In addition, to identify cellular proteins that interact with LMP2, His-tagged versions of LMP2A and LMP2B were stably expressed in HEK 293 cells. The expression of LMP2 in these cells was confirmed. A new approach stable isotope labelling amino acid in cell culture (SILAC) to identify interacting proteins which combined with LC-MS/MS analysis was performed and 10 novel LMP2A interactors and 20 novel LMP2B interactors were identified. All these potential interactions require validation but of particular interest is the possible interaction of DNA methyl transferase 1 (DNMT1) with LMP2A and OB-cadherin with LMP2B. Overall, the studies described in this thesis demonstrate that LMP2A and LMP2B have profound effects on the behaviour of HL cells and SILAC combined with LC-MS/MS is a useful and reliable approach to identify interacting proteins.