Functions of Epstein-Barr Virus LMP2A in Latency

描述（由申请人提供）：超过90%的人群携带ebv潜伏感染细胞。在典型的良性B细胞原发感染后，EBV转变为潜伏状态，在这种状态下，病毒表达很少的病毒转录物以避免免疫识别。这些潜伏感染的细胞被认为是ebv相关淋巴瘤的前体，包括伯基特淋巴瘤、霍奇金淋巴瘤和非霍奇金淋巴瘤。潜伏膜蛋白2A （Latent Membrane protein 2A, LMP2A）是EBV相关肿瘤在潜伏期表达的一种EBV蛋白。LMP2A增强B细胞存活，加速肿瘤发生。本研究的初步数据表明，LMP2A可增强丝裂原诱导的B细胞增殖和IL-10的产生，IL-10是一种促进B细胞增殖和存活的细胞因子。因此，lmp2a介导的IL-10产生的增加可能影响病毒潜伏期的维持和ebv相关淋巴瘤的产生。该提议的假设是LMP2A通过增加B细胞IL-10的产生来增强B细胞的增殖。实验使用表达LMP2A和LMP2A阴性的细胞系，以及来自小鼠LMP2A转基因小鼠模型的B细胞，将验证这一假设。在Aim 1中，我们将使用定量实时RT-PCR、ELISpot和细胞内IL-10染色来证实LMP2A增加了丝裂原诱导的IL-10的产生。在Aim 2中，我们将使用含有野生型LMP2A和突变型LMP2A的细胞系来确定增加IL-10产生所需的LMP2A细胞质尾部残基。此外，我们将使用药理学抑制剂灭活LMP2A靶点，以确定增加IL-10产生所需的蛋白质。最后，在Aim 3中，我们将使用一种中和性抗IL-10抗体和IL-10受体信号转导途径特异性抑制剂灭活IL-10信号通路，以确定lmp2a依赖性IL-10的增加是否介导表达lmp2a的B细胞的过度增殖。了解LMP2A增加B细胞增殖和IL-10产生的机制将促进我们对LMP2A如何维持EBV潜伏期和促进EBV相关癌症的认识。

DESCRIPTION (provided by applicant): Over 90% of the population harbor EBV-latently infected cells. After a typically benign primary infection of B cells, EBV transitions to a latent state in which the virus expresses few viral transcripts to avoid immune recognition. These latently-infected cells are believed to be the precursors for EBV-associated lymphomas, including Burkitt's lymphoma, Hodgkin's and Non-Hodgkin's lymphoma. An EBV protein that is expressed during latency and by EBV-associated tumors is Latent Membrane Protein 2A (LMP2A). LMP2A enhances B cell survival and accelerates tumor onset. Preliminary data for this proposal indicate that LMP2A enhances mitogen-induced B cell proliferation and the production of IL-10, which is a cytokine that promotes B cell proliferation and survival. Therefore, LMP2A-mediated increases in IL-10 production may impact the maintenance of viral latency and the generation of EBV-associated lymphomas. The hypothesis for this proposal is that LMP2A enhances B cell proliferation by increasing B cell IL-10 production. Experiments using both LMP2A-expressing and LMP2A-negative cell lines, in addition to B cells derived from murine LMP2A transgenic mouse models will test this hypothesis. In Aim 1 we will use quantitative Realtime RT-PCR, ELISpot, and intracellular IL-10 staining to confirm that LMP2A increases mitogen-induced IL-10 production. In Aim 2 we will use cell lines containing wildtype LMP2A and mutant forms of LMP2A to determine the residues of the cytoplasmic tail of LMP2A required to increase IL-10 production. Additionally, we will use pharmacologic inhibitors that inactivate targets of LMP2A to determine the proteins required to increase IL-10 production. Finally, in Aim 3 we will inactivate the IL-10 signaling pathway using a neutralizing anti-IL-10 antibody and inhibitors specific for the IL-10 receptor signal transduction pathway to determine if LMP2A-dependent increases in IL-10 mediate the hyperproliferation of LMP2A-expressing B cells. Understanding the mechanisms by which LMP2A increases B cell proliferation and IL-10 production will advance our knowledge of how LMP2A maintains EBV latency and promotes EBV-associated cancers. PUBLIC HEALTH RELEVANCE: Over 90% of the population harbor B cells latently infected with Epstein-Barr Virus (EBV) that is associated with the development of lymphomas, such as Burkitt's lymphoma, Hodgkin's lymphoma and Non-Hodgkin's lymphoma. This proposal will expand our understanding of how EBV increases the production of the cytokine IL-10 to increase proliferation and survival of B cells. Findings from these studies will not only help us to understand the biology of EBV, but also to identify mechanisms by which EBV promotes tumor development.