Epstein-Barr virus (EBV) latent membrane protein (LMP) 2A

Abstract: Epstein-Barr virus (EBV) is a ubiquitous human herpes virus which infects more than 90% of individuals in the population and is causally implicated in lymphoid and epithelial malignancies. Virus invades oropharyngeal mucosa and establishes latency in memory B lymphocytes. Of eleven genes expression in latency, EBV latent membrane protein 2A (LMP2A) is important for maintenance of latency in infected B lymphocytes. This study aims to investigate the biological role of LMP2A in the EBV latent infection of B cells and in epithelial cells, with a special reference to tumorigenesis. LMP 2A is expressed both in peripheral B lymphocytes of normal EBV latently infected individuals, and EBVassociated diseases, such as NPC, HL and mononucleosis. Our studies revealed that LMP2A transcripts along with EBNA1 (U-K splicing) expressed in small, resting high density B lymphocytes of healthy EBV carriers, was detected. These data indicate that LMP2A seems to play a physiologic role in maintenance of viral latency (Paper I). LMP2A transcripts were detected in 17 of 24 (70%) NPC tumour biopsies, together with EBNA1 that was detected in all samples driven by Qp, LMP1 and LMP2B are 90% of NPC (paper II). These results suggest that LMP2A is implicated not only in viral latent biology, but also in oncogenic pathology. We investigated the role of LMP2A in three aspects: LMP2A recruits WW domain-containing ubiquitin -protein ligases (III), effect of LMP2A on hTE RT/telome rase activity (IV) and on oncogenic pathology (V). We show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including A1P4 and KIAA0439, and form physiological complexes with LMP2A in EBV-positive B cells. Consequently, LMP2A enhances Lyn and Syk ubiquitination in vivo and correlates with destabilization of the Lyn tyrosine kinase, however, we have not observed any effect of LMP2A on Syk stability. Thus, LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signalling. In addition, LMP2A itself is also ubiquitinated and degraded through proteasome. Therefore, it is likely that LMP2A utilizes ubiquitin-mediated degradation through the proteasome complex to regulate the strength of its own signal, and to further modulate processes of differentiation, activation or survival of B cells in vivo. Recently, several lines of evidence have suggested that activation of telomerase may be a critical for cell immortalization and transformation, in contrast, it is silent in general due to a stringent repression of hTERT in most human somatic cells. Therefore, our interest has been focused on the potential effect of LMP2A on the expression of hTERT/telomerase activity. We found, although unexpected and paradoxical, that LMP2A expression led to the transcriptional repression of the hTERT gene accompanied by a decreased telomerase activity in epithelial cells. Furthermore, LMP2A, through ITAM motif, was shown to inhibit the hTERT promoter activity in both B cells and epithelial cells. Thus, one possibility is that the LMP2A effect on hTERT is related to its role in suppressing physiologic B-cell activation. (Paper IV). Syk tyrosine kinase plays an instrumental role in the EBV infection of B-lymphocytes. However, In line with several recent reports, we have found that the Syk was expressed in human epithelial cell lines, 5637 (derived from human bladder carcinoma) and TW03 (derived from NPC). LMP2A transfectants in 5637 cells could enhance cell growth and produce tumours in SCID mice. LMP2A, via ITAM motifs, bound to and phosphorylated Syk . This correlated with the capacity of LMP2A to induce cell invasion. LMP2A expression or downregulation of Syk by shRNA Syk resulted in enhance of invasive capacity in both cell types, as a potential tumour suppressor. We show that Syk could directly interact with the ITAM-like motif in integrin a6b4, suggesting a6b4 may be the link between Syk and LMP2A-induced invasive capacity. (paper V). These data suggest that LMP2A may use ubiquitination to regulates its own signal strength and further to influence epithelial cell behaviour such as cell adhesion and motility.