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

项目摘要/摘要 EB病毒(EBV)感染口咽上皮细胞与至少两种类型的喉癌有关 疾病：口腔毛状白斑(OHL)，一种由EBV裂解感染分化上皮引起的舌部病变 细胞，和鼻咽癌(NPC)，一种以潜伏的EBV感染为特征的恶性肿瘤 未分化的上皮细胞。艾滋病患者对裂解性和潜伏性EB病毒感染的控制受到损害， 与正常宿主相比，OHL和NPC的发生率增加。两种EBV即刻早期蛋白， BZLF1(Z)和BRLF1(R)，促进EB病毒从潜伏感染到裂解感染的转换。我们最近的研究表明 显示裂解启动子的DNA甲基化增强了Z，但阻碍了裂解感染的R激活。我们激动人心的 初步数据显示，新描述的表观遗传修饰与胞嘧啶去甲基化有关， 5-羟甲基胞嘧啶(5-HMC)对Z与R诱导裂解EBV的能力有深远的影响 重新激活。尽管上皮细胞分化与EBV裂解重新激活有关，但对此的研究 由于缺乏稳定维持EBV感染的器官型体外模型，这种关系一直受到限制。 我们最近鉴定出一种端粒酶永生化的正常口腔角质形成细胞(NOK)系，它可以稳定地携带EBV 感染，表明EBV+NOK在RAFT培养中经历了分化，裂解的EBV重新激活 发生在分化程度较高的细胞中。到目前为止，EBV+NOK是唯一显示含有 在很大程度上没有甲基化的EBV基因组，以及唯一已知的在R之后重新激活的EBV+细胞株，但不是Z， 表情。因此，EBV+NOK是理解表观遗传机制的独特工具 上皮细胞分化对EBV潜伏感染向裂解感染的转化及EBV潜伏裂解基因的作用 产品会影响受感染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 宿主和病毒基因组在分化过程中发生的表观遗传修饰，并导致裂解 并确定BLIMP1在NOK分化过程中EBV再激活中的作用。以特定的目标 2，我们将检测5-HMC修饰对裂解性和潜伏性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LMP1和LMP2A对上皮细胞分化的影响， 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，我们将 确定鼻咽癌标本中5-HMC修饰的程度，并确定5-HMC途径是否 在鼻咽癌肿瘤中被破坏。我们假设a)EBV基因组的甲基化和5-HMC修饰 控制Z与R，b)上皮细胞分化至少部分地通过 对病毒基因组甲基化和5-HMC修饰的影响，以及c)EBV+鼻咽癌仅发生在 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。拟议的研究将大大增强我们对EBV正常复制方式的理解 在分化的上皮性肿瘤细胞中，病毒潜伏期较长，而在未分化的上皮性肿瘤细胞中病毒潜伏期较长。

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.