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B细胞的典型良性初次感染后，EBV转变为潜伏状态，其中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。在潜伏期期间和由EBV相关肿瘤表达的EBV蛋白是潜伏膜蛋白2A（LMP 2A）。LMP 2A增强B细胞存活并加速肿瘤发作。该提议的初步数据表明，LMP 2A增强有丝分裂原诱导的B细胞增殖和IL-10的产生，IL-10是促进B细胞增殖和存活的细胞因子。因此，LMP 2A介导的IL-10产生增加可能影响病毒潜伏期的维持和EBV相关淋巴瘤的产生。该建议的假设是LMP 2A通过增加B细胞IL-10的产生来增强B细胞增殖。使用LMP 2A表达和LMP 2A阴性细胞系以及源自鼠LMP 2A转基因小鼠模型的B细胞的实验将检验该假设。在目标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LMP 2A增加促分裂原诱导的IL-10产生。在目的2中，我们将使用含有野生型LMP 2A和LMP 2A突变体形式的细胞系来确定增加IL-10产生所需的LMP 2A胞质尾区的残基。此外，我们将使用药理学抑制剂来抑制LMP 2A的靶点，以确定增加IL-10产生所需的蛋白质。最后，在目的3中，我们将使用中和抗IL-10抗体和IL-10受体信号转导途径特异性抑制剂来检测IL-10信号转导途径，以确定IL-10中的LMP 2A依赖性增加是否介导表达LMP 2A的B细胞的过度增殖。了解LMP 2A增加B细胞增殖和IL-10产生的机制将促进我们对LMP 2A如何维持EBV潜伏期和促进EBV相关癌症的认识。 公共卫生相关性：超过90%的人群携带潜伏感染EB病毒（EBV）的B细胞，EB病毒与淋巴瘤（例如伯基特淋巴瘤、霍奇金淋巴瘤和非霍奇金淋巴瘤）的发展相关。这一提议将扩大我们对EBV如何增加细胞因子IL-10的产生以增加B细胞的增殖和存活的理解。这些研究结果不仅有助于我们了解EBV的生物学，还有助于确定EBV促进肿瘤发展的机制。

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.