Epigenetic Regulation of HBV cccDNA Transcription

HBV cccDNA 转录的表观遗传调控

• 批准号: 10404066
• 负责人: Haitao Guo
• 金额: $ 38.74万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-16 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10404066
• 关键词: Address; Adverse effects; Affect; Antiviral Agents; Biology; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Cessation of life; Chromatin; Chromosomes; Chronic Hepatitis B; Circular DNA; Cirrhosis; Clinical; Coupled; DNA; DNA Repair; Dependence; Development; Disease; Drug resistance; Epigenetic Process; Episome; Equilibrium; Exhibits; Genetic Transcription; Genomic Segment; Goals; HMGB1 Protein; Hepatitis B Virus; Hepatocyte; High-Throughput Nucleotide Sequencing; Histones; Individual; Integration Host Factors; Interferon-alpha; Lead; Life Cycle Stages; Light; Liver; Liver Fibrosis; Longevity; Maps; Mediating; Methods; Methylation; Modeling; Modification; Nuclear; Outcome; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Polymerase; Post-Translational Protein Processing; Primary carcinoma of the liver cells; Proteins; Proteomics; Public Health; Quality of life; Reporter; Repression; Research Project Grants; Risk Factors; Role; System; Trans-Activators; Treatment Failure; Validation; Variant; Viral; Viral Genome; Viral Regulatory Proteins; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; analog; bisulfite sequencing; chromatin immunoprecipitation; chromosome conformation capture; chronic infection; comparative; comparative genomics; effective therapy; epigenetic profiling; epigenetic regulation; epigenetic silencing; epigenetic therapy; epigenome; experimental study; loss of function; non-histone protein; novel; promoter; response; stable cell line; therapeutic development; treatment strategy; viral genomics

ABSTRACT This project aims at elucidating the mechanisms underlying epigenetic regulation of hepatitis B virus (HBV) covalently closed circular (ccc) DNA transcription in hepatocytes, focusing on viral X protein (HBx)- mediated epigenetic regulation of HBV cccDNA with involvement and fine balancing of host epigenetic modulators. HBV cccDNA is essential to the virus life cycle, its complete elimination or inactivation during chronic infection is considered critical to a cure but has not been achieved by current antivirals. HBV cccDNA exists in the cell nucleus as an individual minichromosome decorated with histones and non-histone proteins. Elucidating the mechanisms of chromatin compactization of cccDNA and principles of epigenetic regulation of cccDNA episome in its interplay with host factors could allow us to elaborate new antiviral strategies for addressing the unmet clinical need. Among the limited number of HBV-encoded proteins, the viral regulatory protein HBx serves as a multifunctional transactivator of the viral and cellular promoters and has been proven to be a potent epigenetic modifying factor in HBV-infected livers. To further address the role of HBx in cccDNA transcription, we have developed a pair of inducible cccDNA reporter stable cell lines with and without HBx expression, namely HepBHAe82 and HepBHAe∆x67. While both cell lines are able to produce comparable level of cccDNA regardless of the presence or absence of HBx, the cccDNA in HepBHAe∆x67 cells is epigenetically silenced. The HBx-dependent cccDNA transcription has also been recapitulated in wildtype and HBx-minus HBV infected hepatocytes. In this project, by making use of these experimental systems, we will systematically characterize the epigenetic profile variations between transcriptionally active and inactive cccDNA (Aim 1), map the interaction of cccDNA minichromosome with host chromosomes (Aim 2), and identify host epigenetic modulators that regulate cccDNA transcription through comparative proteomic approach, followed by functional validation (Aim 3). In Aim 3, we have already identified HMGB1 as a novel host restriction factor for cccDNA, and will further elucidate the mechanism of HMGB1-mediated epigenetic repression of cccDNA transcription and the interplay between HBx and HMGB1 in cccDNA activation. The accomplishment of this project will shed more light on the cccDNA epigenetics, and provide novel antiviral targets for development of therapeutics that epigenetically silence cccDNA to achieve a functional cure of chronic hepatitis B.

抽象的 该项目旨在阐明丙型肝炎病毒的表观遗传调节的机制 （HBV）在肝细胞中共价闭合圆形（CCC）DNA转录，重点是病毒X蛋白（HBX） - HBV CCCDNA的介导的表观遗传调节，宿主表观遗传学的参与和精细平衡 调节器。 HBV CCCDNA对于病毒生命周期至关重要，其完全消除或在慢性期间失活 感染对治疗至关重要，但尚未通过当前抗病毒药来实现。 HBV CCCDNA存在于 细胞核作为单个微型小体，并用组蛋白和非历史蛋白装饰。阐明 CCCDNA染色质压实的机制和CCCDNA的表观遗传调节原理 在与宿主因素相互作用的相互作用中，情节可以使我们能够制定新的抗病毒策略来解决该策略 未满足的临床需求。在有限数量的HBV编码蛋白中，病毒调节蛋白HBX服务 作为病毒和细胞启动子的多功能反式激活剂，已被证明是有效的 HBV感染生命中的表观遗传修饰因子。为了进一步解决HBX在CCCDNA转录中的作用， 我们已经开发了一对具有和不表达HBX的诱导型CCCDNA记者稳定细胞系，即 hepbhae82和hepbhae支是。虽然这两种细胞系都能够产生可比水平的CCCDNA 不管HBX的存在或不存在，HepbhaeΔX67细胞中的CCCDNA表观遗传沉默。 HBX依赖性CCCDNA转录也已在感染的WildType和HBX-Minus HBV中概括 肝细胞。在这个项目中，通过使用这些实验系统，我们将系统地表征 转录活性和无活性CCCDNA之间的表观遗传曲线变化（AIM 1），绘制相互作用 用宿主染色体（AIM 2）的CCCDNA微型体小体（AIM 2），并识别宿主表观遗传调节剂 通过比较蛋白质组学方法调节CCCDNA转录，然后进行功能验证（目标 3）。在AIM 3中，我们已经将HMGB1确定为CCCDNA的新型宿主限制因子，并将进一步 阐明HMGB1介导的CCCDNA转录和相互作用的表观遗传表达的机制 在CCCDNA激活中HBX和HMGB1之间。该项目的成就将使 CCCDNA表观遗传学，并为开发具有表观遗传学的治疗剂提供新颖的抗病毒靶标 沉默CCCDNA以实现慢性丙型肝炎的功能治愈。