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: 8625514
• 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/8625514
• 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; 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

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.

项目摘要/摘要 口咽上皮细胞的爱泼斯坦 - 巴尔病毒（EBV）感染至少两种类型 疾病：口服毛状白细胞（OHL），这是由分化上皮的裂解EBV感染引起的舌头病变 细胞和鼻咽癌（NPC），一种具有潜在EBV感染为特征的恶性肿瘤 未分化的上皮细胞。艾滋病患者控制裂解和潜在的EBV感染以及 与正常宿主相比，OHL和NPC的发生率增加。两种EBV即时蛋白质， BZLF1（Z）和BRLF1（R），促进了从潜在到裂解EBV感染的转换。我们最近的研究已经 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。我们令人兴奋 初步数据表明，新描述的表观遗传修饰与胞嘧啶脱甲基化有关 5-羟基甲基 - 氰（5-HMC）对Z与R诱导裂解EBV的能力具有深远的影响 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。 我们最近确定了一个永生的正常口服角质形成细胞（NOK）线，可以稳定EBV 感染，表明EBV+ NOKS在筏培养物中经历了分化，并且裂解EBV重新激活 特别发生在更分化的细胞中。迄今为止，EBV+ NOK是显示的唯一包含的单元线 在很大程度上没有甲基化的EBV基因组，并且是唯一已知在R之后重新激活但不是Z的EBV+细胞系 表达。因此，EBV+ NOKS是理解链接的表观遗传机制的独特工具 上皮细胞分化为EBV潜在裂解感染的转化以及EBV潜在和裂解基因 产品影响感染口腔上皮细胞的表观遗传状态。在特定目标1中，我们将描述 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。在特定目标中 2，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。在特定目标4中，我们将 表征NPC样品中5-HMC修饰的程度，并确定5-HMC途径是否为 在NPC肿瘤中破坏。我们假设A）EBV基因组的甲基化和5-HMC修饰 控制Z与R，B）上皮分化的重新激活至少部分通过 对病毒基因组甲基化和5-HMC修饰的影响，c）EBV+ NPC肿瘤仅发生在 未分化的上皮细胞，至少部分通过病毒基因组表观遗传促进病毒潜伏期 修改。拟议的研究应大大增强我们对EBV通常如何复制的理解 在分化的上皮细胞中，但在未分化的上皮肿瘤细胞中达到了长期的病毒潜伏期。