Greater than 90% of the world's population is infected with the human herpes virus Epstein-Barr virus (EBV), which establishes latency in B lymphocytes. In immunocompromised individuals, reactivation of latent EBV in the absence of normal immune control can result in the development or B cell lymphoma. Among the EBV-encoded proteins implicated in lymphomagenesis, considerable attention has focused upon latent membrane protein-1 (LMP1), the only EBV-produced protein that can directly transform cells in culture; LMP1- mutant viruses cannot transform B cells. Our lab studies CD40, a member of the tumor necrosis factor receptor (TNF-R) family expressed on B cells, macrophages, and dendritic cells that induces B cell proliferation, isotype switching, and upregulation of surface molecules involved in antigen presentation. We became very interested in LMP 1 upon learning that this viral protein interacts with certain cytoplasmic adapter proteins (TNF-R associated factors, or TRAFs), previously characterized as only binding to TNF-R family molecules, such as CD40. We found that LMP1 signals in B cells mimic CD40 to a striking extent. However, when we directly compare signaling to B cells by the two molecules, LMP 1 signals occur more rapidly, and are amplified and sustained compared to those delivered through CD40. These differences map to the cytoplasmic (CY) domains of the two molecules, and correlate with the ability of CD40 to induce TRAF degradation, an ability that LMP1 lacks. In the present proposal we wish to determine the molecular basis for differences between CD40 and LMP1 signaling, and how these affect B cell behavior. Our specific goals and the questions to be addressed are as follows:
Aim 1. What is the molecular basis for differences in signaling between LMP 1 and its normal cellular counterpart, CD40? A. What is the role of the reduced binding affinity of TRAF2 for LMP1, compared to CD40, in the failure of LMP1 to induce TRAF2 and 3 degradation? B. Does association of TRAF6 with CD40, but not LMP 1, contribute to signaling differences between the two molecules? C. How do the signaling pathways of LMP 1 and CD40 show differential dependence upon distinct TRAFs? Aim 2. How do signaling differences between LMP1 and CD40 affect the function of B cells in the intact animal? A. What is the phenotype of antigen-presenting cells in mice expressing Wt CD40 versus CD40 with an LMP1 cytoplasmic domain? B. How does the humoral response to T-dependent (TD) and T-independent (TI) antigens compare in WtmCD40tg and mCD40LMP 1tg mice? C. What are the characteristics of TRAF association and regulation with CD40 and CD40-LMP1 in cells from transgenic mice expressing these receptors? D. How do differences in TRAF association between LMP1 and CD40 affect the B cell response? Aim 3. What is the effect of signaling via the LMP 1 cytoplasmic domain on autoimmune responses? A. What is the relationship between LMP 1 signals and autoantibody production? B. How does LMP1 expression affect the development and progression of autoimmune disease?
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