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）：乙型肝炎病毒（HBV）仍然是慢性肝病（包括肝硬化和癌症）的全球病因。目前针对慢性乙型肝炎的抗病毒治疗仅部分有效。特别是，附加型病毒DNA，即所谓的共价闭合环状（CCC）DNA，即使经过多年的抗病毒治疗，仍然存在于受感染的细胞核中。 CCC DNA 是所有病毒转录的模板，也是 HBV 持久性的分子基础。因此，消除 CCC DNA 是治愈 HBV 感染的先决条件。 CCC DNA 由病毒基因组 DNA 产生，具有松弛环状 (RC)、部分双链结构。为了完成 RC 到 CCC DNA 的转化过程，必须发生多种生化反应，目前对此还一无所知。当前应用的总体目标是开始使用 HBV 和鸭 HBV (DHBV) 作为模型系统来分析 CCC DNA 形成的分子机制。提出了三个具体目标。具体目标 1 是确定 CCC DNA 形成的潜在途径，包括假定的中间体。我们计划利用体外细胞培养系统（其中 HBV 和 DHBV CCC DNA 形成并积累潜在中间体）来详细识别和表征这些中间体。这与具体目标 2 和 3 中提出的干扰其产生的定向方法相结合，将为 CCC DNA 形成的潜在途径提供重要线索。具体目标 2 将结合遗传和生化方法，确定特定病毒因子（即病毒包膜和逆转录酶蛋白）在 CCC DNA 形成和调节中的作用。具体目标 3 将使用现有的细胞培养系统和将要开发的无细胞测定法，确定选定的宿主因子，特别是细胞 DNA 修复因子在 CCC DNA 形成中的作用。这些研究将为 CCC DNA 形成机制带来急需的见解，这可能有助于开发直接针对病毒复制这一关键步骤的新型抗病毒药物。此外，它们还可能为细胞 DNA 损伤修复机制提供新的线索，细胞 DNA 损伤修复机制的故障是从发育缺陷到癌症等多种严重人类疾病的基础。公共卫生相关性：乙型肝炎病毒 (HBV) 是导致慢性肝病（包括肝硬化和癌症）的全球性原因。我们建议阐明产生核附加型病毒 DNA 的机制以及参与的病毒和宿主因素，这是 HBV 持久性的分子基础。这些研究应有助于开发直接针对病毒复制这一关键步骤并能够治愈持续性感染的新型抗病毒药物。