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

描述（由申请人提供）：eb病毒（EBV）感染口咽上皮细胞与至少两种类型的疾病相关：口腔毛状白斑（OHL），一种由分化上皮细胞溶解性EBV感染引起的舌头病变，以及鼻咽癌（NPC），一种以未分化上皮细胞潜伏性EBV感染为特征的恶性肿瘤。与正常宿主相比，艾滋病患者对溶解性和潜伏性EBV感染的控制受损，OHL和NPC的发病率增加。两种EBV即时早期蛋白，BZLF1 (Z)和BRLF1 (R)，促进从潜伏型EBV感染到裂解型EBV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对R诱导裂解性EBV再激活的能力有深远的影响。虽然上皮细胞分化与EBV裂解再激活有关，但这方面的研究

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.