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Epigenetic Regulation of HBV cccDNA Transcription

HBV cccDNA 转录的表观遗传调控

基本信息

批准号：
10624470
负责人：
Haitao Guo
金额：
$ 38.76万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/10624470
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 Exhibits Genetic Transcription Genomic Segment Goals HMGB1 gene Hepatitis B Virus Hepatocyte High-Throughput Nucleotide Sequencing Histones Individual Integration Host Factors Interferon alpha Lead Life Cycle Stages 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 Productivity Proteins Proteomics Public Health Quality of life Relaxation 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 therapy epigenome experimental study loss of function non-histone protein novel promoter response stable cell line therapeutic development treatment strategy viral genomics

项目摘要

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乙肝病毒表观遗传调控的机制。肝细胞共价闭合环(CCC)DNA转录，关注病毒X蛋白(HBx)- 宿主表观遗传参与和精细平衡介导的HBVcccDNA表观遗传调控调制器。HBVcccDNA对病毒的生命周期是必不可少的，在慢性疾病过程中，病毒完全消除或失活。感染被认为是治愈的关键，但目前的抗病毒药物并不能实现感染。HBVcccDNA存在于细胞核是一个单独的微小染色体，用组蛋白和非组蛋白修饰。澄清 CCCDNA染色质紧凑化机制及其表观遗传调控原理 Episome与宿主因素的相互作用可能使我们能够制定新的抗病毒策略来解决未得到满足的临床需求。在有限数量的乙肝病毒编码蛋白中，病毒调节蛋白HBx起作用作为病毒和细胞启动子的多功能反式激活剂，已被证明是一种有效的乙肝病毒感染肝脏中的表观遗传修饰因子。为了进一步研究HBx在CCDNA转录中的作用，我们已经建立了一对可诱导的cccDNA报告基因稳定表达和不表达HBx的细胞系，即 HepBHAe 82和HepBHAe∆x67。而两种细胞系都能产生水平相当的cccDNA 无论是否存在HBx，在HepBHAe∆x67细胞中的cccDNA在表观遗传学上是沉默的。在野生型和HBx阴性的乙肝病毒感染中，依赖于HBx的cccdna转录也被重述。肝细胞。在这个项目中，通过利用这些实验系统，我们将系统地表征转录活性和非活性cccDNA之间的表观遗传差异(Aim 1)，绘制了相互作用图 CcCDNA微染色体与宿主染色体的关系(目标2)，并鉴定宿主表观遗传调节子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是ccccDNA的一个新的宿主限制因子，并将进一步阐明HMGB1介导的表观遗传抑制cccDNA转录的机制及其相互作用 HBx和HMGB1在ccDNA激活中的作用。这个项目的完成将使我们更清楚地认识到 CccDNA表观遗传学，并为表观遗传学治疗药物的开发提供新的抗病毒靶点沉默cccDNA以实现慢性乙肝的功能性治疗