Epigenetic Regulation of HBV cccDNA Transcription

HBV cccDNA 转录的表观遗传调控

• 批准号: 10194361
• 负责人: Haitao Guo
• 金额: $ 38.96万
• 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/10194361
• 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 该项目旨在阐明乙型肝炎病毒表观遗传调控的机制 (HBV) 肝细胞中的共价闭合环状 (ccc) DNA 转录，重点关注病毒 X 蛋白 (HBx)- HBV cccDNA 介导的表观遗传调控，涉及宿主表观遗传的参与和精细平衡 HBV cccDNA 调节剂对于病毒的生命周期、慢性过程中的完全消除或失活至关重要。 感染被认为是治愈的关键，但目前的抗病毒药物尚未实现 HBV cccDNA 的治疗。 细胞核作为单个微型染色体，饰有组蛋白和非组蛋白。 cccDNA染色质致密化机制及cccDNA表观遗传调控原理 附加体与宿主因子的相互作用可以让我们制定新的抗病毒策略来解决 在数量有限的 HBV 编码蛋白中，病毒调节蛋白 HBx 服务于未满足的临床需求。 作为病毒和细胞启动子的多功能反式激活子，已被证明是一种有效的 HBV 感染肝脏中的表观遗传修饰因子 为了进一步研究 HBx 在 cccDNA 转录中的作用， 我们开发了一对可诱导的 cccDNA 报告基因稳定细胞系，有和没有 HBx 表达，即 HepBHAe82 和 HepBHAeΔx67 两种细胞系都能够产生相当水平的 cccDNA。 无论是否存在 HBx，HepBHAeΔx67 细胞中的 cccDNA 都会被表观遗传沉默。 HBx 依赖性 cccDNA 转录也在野生型和 HBx-minus HBV 感染中得到重现 在这个项目中，通过利用这些实验系统，我们将系统地表征肝细胞。 转录活性和非活性 cccDNA 之间的表观遗传谱差异（目标 1），绘制相互作用图 cccDNA 微型染色体与宿主染色体的结合（目标 2），并鉴定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（Aim 3). 在目标3中，我们已经确定HMGB1为cccDNA的新宿主限制因子，并将进一步确定。 阐明 HMGB1 介导的 cccDNA 转录表观遗传抑制机制及其相互作用 该项目的完成将为 cccDNA 激活中 HBx 和 HMGB1 之间的关系提供更多线索。 cccDNA 表观遗传学，并为开发表观遗传学疗法提供新的抗病毒靶点 沉默cccDNA以实现慢性乙型肝炎的功能性治愈。