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

描述(申请人提供)：EB病毒感染口咽上皮细胞至少与两种疾病有关：口腔毛状白斑(OHL)，一种由分化的上皮细胞裂解的EBV感染引起的舌部病变；以及鼻咽癌(NPC)，一种以未分化的上皮细胞潜伏的EBV感染为特征的恶性肿瘤。与正常宿主相比，艾滋病患者损害了对裂解性和潜伏性EBV感染的控制，并增加了OHL和NPC的发生率。两种EBV即刻早期蛋白BZLF1(Z)和BRLF1(R)促进了EBV从潜伏感染到裂解感染的转换。我们最近的研究表明，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。我们令人兴奋的初步数据显示，新描述的与胞嘧啶去甲基化有关的表观遗传修饰，5-羟甲基胞嘧啶(5-HMC)，对Z与R诱导裂解EBV重新激活的能力有深远的影响。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。我们最近发现了一个端粒酶永生化的正常口腔角质形成细胞(NOK)可以稳定地感染EBV，证明了EBV NOK在RAFT培养中经历了分化，并且裂解的EBV重新激活发生在更高分化的细胞中，到目前为止，EBV NOK是唯一被证明含有大量未甲基化的EBV基因组的细胞系，也是唯一已知的在R而不是Z表达后重新激活的EBV细胞系。因此，EBV NOKs是一种独特的工具，可以用来理解将上皮细胞分化与EBV潜伏感染向裂解感染转化的表观遗传学机制，以及EBV潜伏和裂解基因产物如何影响感染的口腔上皮细胞的表观遗传状态。在特定的目标1中，我们将表征在分化过程中宿主和病毒基因组的表观遗传修饰，并导致裂解重新激活，并确定BLIMP1在NOK分化过程中重新激活EBV的作用。在特定目的2中，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、病毒复制以及病毒和细胞基因组的表观遗传状态的影响。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途径在鼻咽癌中是否被破坏。我们假设a)EBV基因组的甲基化和5-HMC修饰控制了Z对R的重新激活，b)上皮分化至少部分地通过对病毒基因组甲基化和5-HMC修饰的影响来调节EBV的重新激活，以及c)EBV鼻咽癌只发生在未分化的上皮细胞中，这些细胞至少部分地通过病毒基因组的表观遗传修饰来促进病毒潜伏。拟议的研究将大大提高我们对EBV如何在分化的上皮细胞中正常复制，但在未分化的上皮细胞中实现长期病毒潜伏的理解 肿瘤细胞。

DESCRIPTION (provided by applicant): 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.