Epigenetic determinants of Epstein-Barr virus and cellular DNA in oral diseases

项目总结/摘要 口咽上皮细胞的EB病毒（EBV）感染与至少两种类型的炎症相关。 疾病：口腔毛状白斑（OHL），一种由分化上皮细胞的溶解性EBV感染引起的舌病变， 细胞和鼻咽癌（NPC），一种以潜伏性EBV感染为特征的恶性肿瘤， 未分化的上皮细胞艾滋病患者对溶解性和潜伏性EBV感染的控制受损， 与正常宿主相比，OHL和NPC的发病率增加。两种EBV立即早期蛋白， BZLF 1（Z）和BRLF 1（R）促进潜伏性EBV感染向裂解性EBV感染的转变。我们最近的研究 显示裂解启动子的DNA甲基化增强Z，但阻碍裂解感染的R活化。整理的丰厚 初步数据显示新描述的表观遗传修饰涉及胞嘧啶去甲基化， 5-羟甲基-胞嘧啶（5-hmC）对Z与R诱导裂解性EBV的能力有深远的影响 重新激活虽然上皮细胞分化与EBV裂解性再活化有关，但对这一点的研究表明， 由于缺乏稳定维持EBV感染的器官型体外模型，这种关系受到限制。 我们最近鉴定了一个端粒酶永生化的正常口腔角化细胞（NOK）系， 感染，表明EBV+ NOKs在筏培养物中经历分化，并且裂解性EBV再活化 特别是在分化程度更高的细胞中。迄今为止，EBV+ NOK是唯一显示含有 大部分未甲基化的EBV基因组，以及已知在R之后而不是Z之后再活化的唯一EBV+细胞系， 表情因此，EBV+ NOKs是理解与EBV + NOKs相关的表观遗传机制的独特工具。 上皮细胞分化为潜伏型向裂解型转化的EBV感染及EBV潜伏和裂解基因是如何表达的 产品影响受感染的口腔上皮细胞的表观遗传状态。在具体目标1中，我们将描述 宿主和病毒基因组的表观遗传修饰发生在分化过程中，并导致裂解 重新激活，并确定BLIMP 1在NOK分化过程中重新激活EBV中的作用。具体目标 2.我们将检测5-hmC修饰对裂解型和潜伏型EBV基因表达的影响。在具体目标3中， 我们将使用EBV+ NOK系统来检测LMP 1和LMP 2A对上皮细胞分化的作用， 病毒复制以及病毒和细胞基因组的表观遗传状态。在具体目标4中，我们 表征NPC标本中5-hmC修饰的程度，并确定5-hmC途径是否 在NPC肿瘤中被破坏。我们假设a）EBV基因组的甲基化和5-hmC修饰 控制Z对R的再活化，B）上皮分化至少部分地通过以下途径调节EBV再活化： 对病毒基因组甲基化和5-hmC修饰的影响，以及c）EBV+ NPC肿瘤仅发生在 至少部分通过病毒基因组表观遗传促进病毒潜伏的未分化上皮细胞 修改.这项研究将极大地提高我们对EB病毒如何正常复制的理解 在分化的上皮细胞中，但在未分化的上皮肿瘤细胞中实现了长期的病毒潜伏期。

PROJECT SUMMARY/ABSTRACT Epstein-Barr virus (EBV) infection of oropharyngeal epithelial cells is associated with at least two types of disease: oral hairy leukoplakia (OHL), a tongue lesion caused by lytic EBV infection of differentiated epithelial cells, and nasopharyngeal carcinoma (NPC), a malignancy characterized by latent EBV infection of undifferentiated epithelial cells. AIDS patients have impaired control of lytic and latent EBV infection and increased incidence of OHL and NPC compared to normal hosts. The two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), promote the switch from latent to lytic EBV infection. Our recent studies have shown DNA methylation of lytic promoters enhances Z, but impedes R activation of lytic infection. Our exciting preliminary data reveal that the newly described epigenetic modification implicated in cytosine demethylation, 5-hydroxymethyl-cytosine (5-hmC), has a profound effect upon the ability of Z versus R to induce lytic EBV reactivation. Although epithelial cell differentiation has been linked to EBV lytic reactivation, studies of this relationship have been limited by the lack of an organotypic in vitro model that stably maintains EBV infection. We recently identified a telomerase immortalized normal oral keratinocyte (NOK) line that can be stably EBV infected, demonstrated that EBV+ NOKs undergo differentiation in raft cultures, and that lytic EBV reactivation occurs specifically in the more differentiated cells. To date, EBV+ NOK is the only cell line shown to contain largely unmethylated EBV genomes, and the only EBV+ cell line known to reactivate following R, but not Z, expression. Thus, EBV+ NOKs are a unique tool for understanding the epigenetic mechanisms that link epithelial cell differentiation to the conversion of EBV latent to lytic infection and how EBV latent and lytic gene products influence the epigenetic state of infected oral epithelial cells. In Specific Aim 1, we will characterize the epigenetic modifications of the host and viral genomes that occur during differentiation, and result in lytic reactivation and determine the role of BLIMP1 in reactivating EBV during NOK differentiation. In Specific Aim 2, we will examine the effect of 5-hmC modification on lytic and latent EBV gene expression. In Specific Aim 3, we will use the EBV+ NOK system to examine the effects of LMP1 and LMP2A on epithelial cell differentiation, viral replication, and the epigenetic state of the viral and cellular genomes. In Specific Aim 4, we will characterize the extent of 5-hmC modification in NPC specimens and determine if the 5-hmC pathway is disrupted in NPC tumors. We hypothesize that a) methylation and 5-hmC modification of the EBV genome controls reactivation by Z versus R, b) epithelial differentiation regulates EBV reactivation at least partially via effects on viral genome methylation and 5-hmC modification, and c) EBV+ NPC tumors only occur in undifferentiated epithelial cells that promote viral latency at least partially via viral genome epigenetic modifications. The proposed studies should greatly enhance our understanding of how EBV normally replicates in differentiated epithelial cells yet achieves long term viral latency in undifferentiated epithelial tumor cells.