text: Introduction
  Epstein-Barr virus (EBV) is a ubiquitous gamma-herpesvirus that infects humans predominantly at an early age with greater than 90% of the adult population infected with EBV [1]. EBV is linked to the development of both B lymphocyte and epithelial cell malignancies, including Burkitt lymphoma, Hodgkin disease (HD), and nasopharyngeal carcinoma (NPC), and cancers linked to immunosuppression, including post-transplant lymphoma and AIDS-associated lymphomas [2,3]. In vitro infection of B lymphocytes with EBV induces permanent growth transformation, and this ability to affect cell growth regulation likely contributes to the development of cancer. 
  Many of the viral proteins expressed in transformed cells, including the EBV nuclear antigens and latent membrane proteins, have profound effects on cell growth regulation and are required for EBV latent infection and B cell transformation [1]. Latent membrane protein 1 (LMP1) is considered the major oncoprotein of EBV, as it transforms rodent fibroblasts to tumorigenicity in nude mice and is expressed in HD, NPC, and immunosuppression-associated tumors [4-8]. In B lymphocytes, LMP1 mimics CD40 signaling, and both LMP1 and CD40 are essential for EBV-mediated B cell transformation [9-11]. While CD40 interacts with CD40 ligand expressed on activated T cells to induce B cell activation and differentiation, LMP1 acts as a constitutive signal through ligand-independent oligomerization. LMP1 and CD40 interact with the same tumor necrosis factor receptor-associated factors (TRAFs) leading to activation of NFkappaB, c-Jun N terminal kinase (JNK), and p38 MAPK signaling pathways [12-16]. Activation of NFkappaB is required for EBV-induced B cell transformation and its inhibition rapidly results in cell death [17,18]. Recent studies indicate that LMP1 also activates phosphatidylinositol 3 kinase (PI3K)/Akt signaling and that this activation is required for LMP1-mediated transformation of rodent fibroblasts [5,19]. 
  In vitro, primary B cells can be maintained by CD40 ligation in combination with IL4 treatment. In vivo, CD40 signaling is necessary for germinal center (GC) formation such that mice deficient for CD40 or CD40L are unable to form GCs in response to T cell-dependent antigens [20,21]. Both the membrane proximal and distal cytoplasmic regions of CD40 that bind TRAF6 and TRAFs2/3/5, respectively, are necessary for GC formation, but either region is sufficient to induce extrafollicular B cell differentiation and restore low affinity antibody production [22]. Functionally, LMP1 can rescue CD40-deficient mice and restore immunoglobulin (Ig) class switching, most likely because LMP1 recruits similar TRAF molecules, TRAFs 1/2/3/5 and TRAF6, through the C-terminal activation regions 1 and 2 domains, respectively. However, LMP1 is unable to restore affinity maturation and GC formation [23]. 
  Several EBV transforming proteins have been studied in transgenic mouse models, however, only LMP1 induces tumor development when expressed under the control of the Ig heavy chain promoter and enhancer [24-26]. The LMP1 transgenic mice (IgLMP1) express LMP1 in B lymphocytes, and in mice older than 12 mo, lymphoma develops with increased incidence (40%-50%) compared to wild-type control mice (11%), suggesting that LMP1 contributes to tumor development [26]. The LMP1 lymphomas have rearranged Ig genes and have activated Akt, JNK, p38, and NFkappaB, with specific activation of the NFkappaB family member cRel [27]. 
  In this study, the LMP1 transgenic lymphocytes and lymphomas were further characterized and their growth properties in vitro were determined. To obtain pure populations of malignant lymphocytes and to enable more detailed biochemical analyses, examples of primary lymphomas were inoculated and passaged in SCID mice. Interestingly, lymphoma development was restricted to B-1a lymphocytes, a self-replenishing population of cells that are prone to malignancy [28,29]. LMP1 transgenic lymphocytes had increased viability in vitro and viability was increased by the addition of IL4. In contrast, both LMP1-positive and -negative lymphoma cells were independent of IL4 co-stimulation for survival and proliferation in vitro with a complete absence of activated Stat6, the IL4 target. The lymphomas were also distinguished by constitutive activation of Stat3 and deregulation of the Rb cell cycle pathway. Inhibition of the PI3K/Akt, NFkappaB, and Stat3 signaling pathways blocked the enhanced growth of both LMP1 transgenic and malignant lymphocytes, suggesting that these pathways are required for their growth and survival. These appear to be the same targets that are deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. This study reveals that LMP1 promotes malignancy in cells with the inherent ability to proliferate and that the Akt, NFkappaB, and Stat3 signaling pathways are required for its growth stimulatory effects. 
  