B cell signaling by the EBV transforming protein, LMP1

描述（由申请人提供）：世界上超过90%的人口感染了人类疱疹病毒eb病毒（EBV），该病毒在B淋巴细胞中建立潜伏期。在免疫功能低下的个体中，在缺乏正常免疫控制的情况下，潜伏性EBV的再激活可导致B细胞淋巴瘤的发展。在eb病毒编码的与淋巴瘤发生有关的蛋白中，相当多的注意力集中在潜伏膜蛋白-1 （LMP1）上，这是eb病毒产生的唯一一种在培养中可以直接转化细胞的蛋白；LMP1突变型病毒不能转化B细胞。CD40是肿瘤坏死因子受体（TNF-R）家族的一员，在B细胞、巨噬细胞和树突状细胞上表达，诱导B细胞增殖、同型转换和参与抗原呈递的表面分子的上调。在了解到这种病毒蛋白与某些细胞质适配器蛋白（TNF-R相关因子，或TRAFs）相互作用后，我们对lmp1非常感兴趣，这些蛋白以前被认为只与TNF-R家族分子结合，如CD40。我们发现B细胞中的LMP1信号在很大程度上模仿CD40。然而，当我们直接比较这两种分子传递给B细胞的信号时，与通过CD40传递的信号相比，lmp1信号发生得更快，并且被放大和持续。这些差异映射到两个分子的细胞质（CY）结构域，并与CD40诱导TRAF降解的能力相关，而LMP1缺乏这种能力。在目前的提议中，我们希望确定CD40和LMP1信号传导差异的分子基础，以及它们如何影响B细胞的行为。我们的具体目标和需要解决的问题如下：

DESCRIPTION (provided by applicant): 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?