Molecular Mechanism of Hepadnavirus Persistence

嗜肝DNA病毒持久性的分子机制

• 批准号: 7777354
• 负责人: Jianming Hu
• 金额: $ 30.06万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777354
• 关键词: Affect; Antiviral Agents; Antiviral Therapy; Biochemical; Biochemical Pathway; Biochemical Reaction; Biological Assay; Biological Models; Cell Culture System; Cell Extracts; Cell Nucleus; Cell-Free System; Cells; Chronic; Chronic Hepatitis B; Chronic viral hepatitis; Circular DNA; Cirrhosis; Conversion disorder; Coupled; DNA; DNA Repair; DNA repair protein; Defect; Development; Duck Hepatitis B Virus; Ducks; Excision; Family; Gene Expression; Genetic; Genetic Transcription; Genome; Genomics; Goals; Hepadnaviridae; Hepatitis B Virus; Hepatocyte; In Vitro; Infection; Integration Host Factors; Ligation; Light; Link; Liver Cirrhosis; Liver diseases; Malignant Neoplasms; Membrane Proteins; Molecular; Mutagenesis; Nuclear; Nucleocapsid; Pathway interactions; Primary carcinoma of the liver cells; Process; Production; Protein Analysis; Proteins; RNA; RNA Caps; RNA-Directed DNA Polymerase; Regulation; Relax transcriptional regulator; Research; Resistance; Retroviridae; Reverse Transcription; Risk; Role; Simian B disease; Staging; Structure; Testing; Viral; Viral Genes; Viral Reverse Transcription; Virion; base; combinatorial; ds-DNA; homologous recombination; human disease; insight; multicatalytic endopeptidase complex; novel; pgRNA; public health relevance; reconstitution; repaired; terminal redundancy; viral DNA

DESCRIPTION (provided by applicant): The hepatitis B virus (HBV) remains a global cause of chronic liver diseases, including liver cirrhosis and cancer. Current antiviral therapy for chronic hepatitis B is only partially effective. In particular, the episomal viral DNA, the so-called covalently closed circular (CCC) DNA, persists in the infected cell nucleus even after years of antiviral treatment. The CCC DNA serves as the template for all viral transcriptions and is the molecular basis of HBV persistence. Therefore, the elimination of the CCC DNA is a prerequisite for any curing of an HBV infection. The CCC DNA is generated from the viral genomic DNA, which has a relaxed circular (RC), partially double-stranded structure. To complete the RC to CCC DNA conversion process, multiple biochemical reactions have to occur, about which nothing is currently understood. The overall goal of the current application is to begin to analyze the molecular mechanisms of CCC DNA formation, using both HBV and the duck HBV (DHBV) as model systems. Three Specific Aims are proposed. Specific Aim 1 will be to determine the potential pathways, including putative intermediates, of CCC DNA formation. Using in vitro cell culture systems where HBV and DHBV CCC DNA formation takes place and potential intermediates accumulate, we plan to identify and characterize these intermediates in detail. This, coupled with directed approaches to perturb their production as proposed in Specific Aims 2 & 3, will provide important clues about the potential pathways of CCC DNA formation. Specific Aim 2 will determine the role of specific viral factors, i.e., the viral envelope and reverse transcriptase proteins, in the formation and regulation of CCC DNA, employing a combination of genetic and biochemical approaches. Specific Aim 3 will determine the role of selected host factors, particularly cellular DNA repair factors, in CCC DNA formation, using both existing cell culture systems and cell-free assays that will be developed. These studies should bring much needed insights into the mechanism of CCC DNA formation, which may facilitate the development of novel antivirals targeted directly at this critical step of viral replication. In addition, they may shed new light on the mechanisms of cellular DNA damage repair, the malfunction of which underlies a variety of serious human diseases from developmental defects to cancer. PUBLIC HEALTH RELEVANCE: The hepatitis B virus (HBV) is a global cause of chronic liver diseases, including liver cirrhosis and cancer. We propose to elucidate the mechanisms of, and viral and host factors involved in, producing the nuclear episomal viral DNA, which is the molecular basis of HBV persistence. These studies should facilitate the development of novel antiviral agents targeted directly at this critical step of viral replication and capable of curing persistent infections.

描述（由申请人提供）：丙型肝炎病毒（HBV）仍然是慢性肝病的全球原因，包括肝硬化和癌症。当前用于慢性丙型肝炎的抗病毒疗法仅部分有效。特别是，即使经过多年的抗病毒治疗，即使在感染的细胞核中，所谓的共价闭合（CCC）DNA的共价闭合（CCC）DNA仍然存在。 CCC DNA用作所有病毒转录的模板，是HBV持久性的分子基础。因此，消除CCC DNA是任何固化HBV感染的先决条件。 CCC DNA是由病毒基因组DNA产生的，该病毒基因组DNA具有松弛的圆形（RC），部分双链结构。为了完成RC到CCC DNA转化过程，必须发生多种生化反应，目前尚未了解。当前应用的总体目标是开始使用HBV和Duck HBV（DHBV）作为模型系统分析CCC DNA形成的分子机制。提出了三个具体目标。具体目的1将是确定CCC DNA形成的潜在途径，包括推定的中间体。使用HBV和DHBV CCC DNA形成的体外细胞培养系统并积累了潜在的中间体，我们计划详细识别和表征这些中间体。这是特定目标2和3中提出的定向方法，以驱散其生产，将为CCC DNA形成的潜在途径提供重要的线索。具体目标2将确定特定病毒因子的作用，即病毒包膜和逆转录酶蛋白在CCC DNA的形成和调节中，采用遗传和生化方法的组合。特定的目标3将使用现有的细胞培养系统和将开发的无细胞培养系统和无细胞的测定法确定选定宿主因素，尤其是细胞DNA修复因子的作用。这些研究应为CCC DNA形成的机理带来急需的见解，这可能有助于直接在病毒复制的关键步骤中发展新型抗病毒药物的发展。此外，它们可能会对细胞DNA损伤修复的机制开发新的启示，其故障是从发育缺陷到癌症的各种严重人类疾病的基础。公共卫生相关性：丙型肝炎病毒（HBV）是慢性肝病的全球原因，包括肝硬化和癌症。我们建议阐明产生核偶发病毒DNA的机制，病毒和宿主因素的机制，这是HBV持久性的分子基础。这些研究应促进直接针对病毒复制的关键步骤并能够治愈持续感染的新型抗病毒剂的发展。