B cell signaling by the EBV transforming protein, LMP1

描述(申请人提供)：超过90%的世界人口感染人类疱疹病毒爱泼斯坦-巴尔病毒(EBV)，它建立在B淋巴细胞中的潜伏期。在免疫受损的个体中，在缺乏正常免疫控制的情况下，潜伏的EBV重新激活可能会导致B细胞淋巴瘤的发展。在EBV编码的与淋巴肿大有关的蛋白质中，潜伏膜蛋白-1(LMP1)是唯一一种EBV产生的蛋白质，可以在培养中直接转化细胞；LMP1突变病毒不能转化B细胞。本实验室研究CD40，肿瘤坏死因子受体家族成员，表达在B细胞、巨噬细胞和树突状细胞上，诱导B细胞增殖、同型转换和参与抗原呈递的表面分子上调。当我们了解到LMP 1与某些细胞质适配蛋白(肿瘤坏死因子相关因子，或TRAF)相互作用后，我们对LMP 1产生了浓厚的兴趣，而以前的特征是只与肿瘤坏死因子家族的分子结合，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个分子对B细胞的信号时，LMP 1信号发生得更快，而且与通过CD40传递的信号相比，LMP 1信号被放大和维持。这些差异映射到这两个分子的细胞质(CY)结构域，并与CD40诱导TRAF降解的能力相关，而LMP1缺乏这一能力。在目前的提案中，我们希望确定CD40和LMP1信号之间的差异的分子基础，以及这些信号如何影响B细胞的行为。我们的具体目标和要解决的问题如下： 目的1.LMP-1与其正常细胞对应分子CD40在信号转导方面存在差异的分子基础是什么？ 与CD40相比，TRAF2与LMP1的结合亲和力降低在LMP1未能诱导TRAF2和TRAF3降解中起什么作用？ TRAF6与CD40的结合，而不是LMP 1的结合，是否有助于这两个分子之间的信号差异？ LMP-1和CD40的信号通路如何对不同的TRAF表现出不同的依赖性？ 目的2.LMP1和CD40之间的信号差异如何影响完整动物的B细胞功能？ 答：在表达Wt CD40和带有LMP1胞浆区的CD40的小鼠中，抗原提呈细胞的表型是什么？ B.在WtmCD40tg和mCD40LMP 1TG小鼠中，对T依赖(TD)和T非依赖(TI)抗原的体液反应如何？ 在表达CD40和CD40-LMP1受体的转基因小鼠的细胞中，TRAF与CD40和CD40-LMP1的结合和调节有什么特征？ LMP1和CD40之间TRAF关联的差异如何影响B细胞的反应？ 目的3.通过LMP-1胞浆结构域传递的信号对自身免疫反应有什么影响？ LMP-1信号和自身抗体的产生有什么关系？ B.LMP1的表达如何影响自身免疫性疾病的发生和发展？

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?