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.

摘要 本项目旨在阐明B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，重点是病毒X蛋白（HBx）- HBV cccDNA介导的表观遗传调控与宿主表观遗传调控的参与和精细平衡 调制器。HBV cccDNA对于病毒生命周期至关重要，其在慢性感染期间完全消除或失活。 感染被认为是治愈的关键，但目前的抗病毒药物还没有实现。HBV cccDNA存在于 细胞核作为一个单独的微小染色体装饰与组蛋白和非组蛋白蛋白。阐明 cccDNA的染色质致密化机制和cccDNA的表观遗传调控原理 附加体与宿主因子的相互作用可以使我们制定新的抗病毒策略， 未满足的临床需求。在有限数量的HBV编码的蛋白质中，病毒调节蛋白HBx充当 作为病毒和细胞启动子的多功能反式激活因子， HBV感染肝脏中的表观遗传修饰因子。为了进一步阐明HBx在cccDNA转录中的作用， 我们已经开发了一对具有和不具有HBx表达的可诱导cccDNA报告基因稳定细胞系，即 HepBHAe 82和HepBHAe 10 x67。虽然两种细胞系都能够产生相当水平的cccDNA， 无论HBx的存在与否，HepBHAe p2 x67细胞中的cccDNA在表观遗传学上是沉默的。 HBx依赖性cccDNA转录也在野生型和HBx阴性HBV感染者中重现 肝细胞在这个项目中，我们将利用这些实验系统， 转录活性和非活性cccDNA之间的表观遗传谱变化（Aim 1），绘制相互作用 的cccDNA微型染色体与宿主染色体（目标2），并确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（目的 3）。在目标3中，我们已经鉴定HMGB 1为cccDNA的新宿主限制因子，并且将进一步 阐明HMGB 1介导的cccDNA转录的表观遗传抑制的机制以及 HBx和HMGB 1在cccDNA激活中的作用。该项目的完成将使人们更清楚地了解 cccDNA表观遗传学，并为开发表观遗传学上 沉默cccDNA以实现慢性B型肝炎功能性治愈。