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: 9318496
• 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 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9318496
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Alpha Cell; B-Lymphocytes; BZLF1 gene; Biopsy; Cell Differentiation process; Cell Line; Cells; ChIP-seq; Characteristics; Chromatin; Cytosine; DNA; DNA Methylation; Data; Development; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Epstein-Barr Virus Infections; Epstein-Barr Virus latency; Frequencies; Gene Activation; Gene Expression; Genes; Genome; Hairy Leukoplakia; Herpesviridae; Human; Human Herpesvirus 4; Immediate-Early Proteins; Impairment; Incidence; Infection; Knowledge; LMP1; Learning; Lesion; Link; Lytic; Lytic Phase; Lytic Virus; Malignant Neoplasms; Maps; Mediating; Methylation; Modeling; Modification; Mouth Diseases; Nasopharynx Carcinoma; Oral; Oropharyngeal; PRDM1 gene; Pathway interactions; Patients; Proteins; Retroviral Vector; Role; Specimen; System; Telomerase; Tongue; Undifferentiated; Viral; Viral Genome; Virus; Virus Diseases; Virus Latency; Virus Replication; demethylation; epigenomics; gain of function mutation; gene product; in vitro Model; keratinocyte; keratinocyte differentiation; knock-down; latent gene expression; latent infection; lytic gene expression; neoplastic cell; novel therapeutics; promoter; public health relevance; response; small hairpin RNA; tool; transcription factor; transcriptome sequencing; tumor; viral transmission

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）：口咽上皮细胞的爱泼斯坦 - 巴尔病毒（EBV）感染至少两种疾病：口服毛状白血病（OHL），这种舌头病变是由裂纹EBV引起的，这是由分化的上皮细胞和鼻cho脑脑（NASOPHARYNGELECELCIFCONTIFE）的裂解EBV引起的，一种舌头感染（NASORCHARYNGELNGEAL CARINDIFCONT），一种，一种舌头。上皮细胞。与正常宿主相比，艾滋病患者对裂解和潜在EBV感染的控制受损，OHL和NPC的发病率增加。两种EBV即时蛋白质BZLF1（Z）和BRLF1（R）促进了从潜在的EBV感染转向。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，新描述的表观遗传学修饰与胞嘧啶脱甲基化有关的5-羟基甲基 - 胞嘧啶（5-HMC）对Z与R诱导Lytic EBV耐激活的能力产生了深远的影响。尽管上皮细胞分化与EBV裂解重新激活有关，但对此进行了研究 由于缺乏稳定维持EBV感染的体外模型，关系受到限制。 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遵循r，但不是z的表达。因此，EBV+ NOKS是理解将上皮细胞分化与EBV潜伏感染与裂解感染的转化以及EBV潜在和裂解基因产物如何影响感染口腔上皮细胞的表观遗传状态的独特工具。在特定目标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，并导致裂解重新激活，并确定Blimp1在NOK分化过程中重新激活EBV中的作用。在特定目标2中，我们将研究5-HMC修饰对裂解和潜在EBV基因表达的影响。在特定的目标3中，我们将使用EBV+ NOK系统来检查LMP1和LMP2A对病毒和细胞基因组的上皮细胞分化，病毒复制以及表观遗传状态的影响。在特定目标4中，我们将 表征NPC标本中5-HMC修饰的程度，并确定在NPC肿瘤中是否破坏了5-HMC途径。 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修改。拟议的研究应大大增强我们对EBV如何在分化的上皮细胞中通常复制但在未分化上皮的长期病毒潜伏期中如何复制的理解 肿瘤细胞。