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的EBV的再活化可导致B细胞淋巴瘤的发展。在EBV编码的与淋巴瘤发生有关的蛋白质中，相当多的注意力集中在潜伏膜蛋白-1（LMP 1）上，LMP 1是EBV产生的唯一可以在培养中直接转化细胞的蛋白质; LMP 1突变病毒不能转化B细胞。我们的实验室研究CD 40，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，同种型转换，并上调参与抗原呈递的表面分子。在了解到这种病毒蛋白与某些细胞质衔接蛋白（TNF-R相关因子或TRAF）相互作用后，我们对LMP 1非常感兴趣，这些蛋白以前仅与TNF-R家族分子（如CD 40）结合。我们发现B细胞中的LMP 1信号在很大程度上模拟了CD 40。然而，当我们直接比较两种分子对B细胞的信号传导时，与通过CD 40递送的信号相比，LMP 1信号发生得更快，并且被放大和维持。这些差异映射到两种分子的胞质（CY）结构域，并与CD 40诱导TRAF降解的能力相关，这是LMP 1缺乏的能力。在目前的建议中，我们希望确定CD 40和LMP 1信号之间差异的分子基础，以及这些差异如何影响B细胞的行为。我们的具体目标和需要解决的问题如下： 目标1。LMP 1与其正常细胞对应物CD 40之间信号传导差异的分子基础是什么？ A.与CD 40相比，TRAF 2与LMP 1的结合亲和力降低，在LMP 1未能诱导TRAF 2和3降解中起什么作用？ B。TRAF 6与CD 40而不是LMP 1的结合是否有助于两种分子之间的信号差异？ C. LMP 1和CD 40的信号通路如何对不同的TRAF表现出不同的依赖性？ 目标二。LMP 1和CD 40之间的信号差异如何影响完整动物中B细胞的功能？ A.在表达Wt CD 40和具有LMP 1胞质结构域的CD 40的小鼠中，抗原呈递细胞的表型是什么？ B。在WtmCD 40 tg和mCD 40 LMP 1 tg小鼠中，对T依赖性（TD）和T非依赖性（TI）抗原的体液反应如何比较？ C.在表达这些受体的转基因小鼠的细胞中，TRAF与CD 40和CD 40-LMP 1的结合和调节的特征是什么？ D. LMP 1和CD 40之间TRAF相关性的差异如何影响B细胞应答？ 目标3。通过LMP 1胞质结构域的信号传导对自身免疫反应有什么影响？ A. LMP 1信号和自身抗体产生之间的关系是什么？ B。LMP 1表达如何影响自身免疫性疾病的发生和进展？

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?