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

口腔疾病中 Epstein-Barr 病毒和细胞 DNA 的表观遗传决定因素

• 批准号: 8737880
• 负责人: ERIC C JOHANNSEN
• 金额: $ 57.89万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8737880
• 关键词: Acquired Immunodeficiency Syndrome; Affect; B-Lymphocytes; BZLF1 protein, Herpesvirus 4, Human; Biopsy; Cell Differentiation process; Cell Line; Cells; ChIP-seq; Characteristics; Chromatin; Cytosine; DNA; DNA Methylation; Data; Development; Disease; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Epstein-Barr Virus Infections; Frequencies; Gene Activation; Gene Expression; Genes; Genome; Hairy Leukoplakia; Herpesviridae; Human; Human Herpesvirus 4; Immediate-Early Proteins; Incidence; Infection; Knowledge; LMP1; Learning; Lesion; Link; Lytic; Lytic Phase; Malignant Neoplasms; Maps; Mediating; Methylation; Modeling; Modification; Mouth Diseases; Nasopharynx Carcinoma; Oral; Oropharyngeal; PRDM1 gene; Pathway interactions; Patients; Proteins; Relative (related person); Retroviral Vector; Role; Specimen; System; Telomerase; Tongue; Undifferentiated; Viral; Viral Genes; Viral Genome; Virus; Virus Latency; demethylation; epigenomics; gain of function mutation; in vitro Model; keratinocyte; keratinocyte differentiation; knock-down; latent gene expression; latent infection; lytic gene expression; neoplastic cell; promoter; public health relevance; response; small hairpin RNA; tool; transcription factor; transcriptome sequencing; transmission process; tumor

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.

描述（由申请人提供）：口咽上皮细胞的EB病毒（EBV）感染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的溶解性EBV感染引起的舌病变，以及鼻咽癌（NPC），一种以未分化上皮细胞的潜伏性EBV感染为特征的恶性肿瘤。与正常宿主相比，AIDS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表明，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），具有深远的影响后，Z与R诱导裂解EBV再激活的能力。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。我们最近鉴定了可被EBV稳定感染的端粒酶永生化的正常口腔角质形成细胞（NOK）系，证明了EBV+ NOK在筏培养物中经历分化，并且裂解性EBV再活化特异性地发生在更分化的细胞中。迄今为止，EBV+ NOK是唯一显示含有大量未甲基化的EBV基因组的细胞系，并且是唯一已知在R而不是Z之后再活化的EBV+细胞系，表情因此，EBV+ NOKs是一种独特的工具，用于理解上皮细胞分化与EBV潜伏性感染转化为裂解性感染的表观遗传机制，以及EBV潜伏性和裂解性基因产物如何影响感染的口腔上皮细胞的表观遗传状态。在具体目标1中，我们将表征分化过程中发生的宿主和病毒基因组的表观遗传修饰，并导致裂解再活化，并确定BLIMP 1在NOK分化过程中再活化EBV中的作用。在具体目标2中，我们将研究5-hmC修饰对裂解和潜伏EBV基因表达的影响。在具体目标3中，我们将使用EBV+ NOK系统来检查LMP 1和LMP 2A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 表征NPC标本中5-hmC修饰的程度，并确定NPC肿瘤中5-hmC通路是否被破坏。我们假设a）EBV基因组的甲基化和5-hmC修饰控制Z与R的再活化，B）上皮分化至少部分地通过对病毒基因组甲基化和5-hmC修饰的影响调节EBV再活化，和c）EBV+ NPC肿瘤仅发生在未分化的上皮细胞中，所述未分化的上皮细胞至少部分地通过病毒基因组表观遗传修饰促进病毒潜伏。这些研究将极大地提高我们对EB病毒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