Regulation of Hepatitis B Virus Capsid Assembly

乙型肝炎病毒衣壳组装的调控

• 批准号: 9761828
• 负责人: Jianming Hu
• 金额: $ 38.58万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761828
• 关键词: Antiviral Agents; Arginine; Bacteria; Binding; C-Peptide; C-terminal; CDK2 gene; Capsid; Capsid Proteins; Cell Extracts; Cell-Free System; Cells; Chronic; Chronic viral hepatitis; Cirrhosis; Complex; Core Protein; Cyclins; DNA; DNA Binding; DNA Viruses; Development; Genome; Hepatitis; Hepatitis B Virus; In Vitro; Insecta; Integration Host Factors; Life Cycle Stages; Liver Cirrhosis; Liver diseases; Malignant neoplasm of liver; Mammalian Cell; Mediating; Methods; N-terminal; Nucleocapsid; Oryctolagus cuniculus; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protamines; Protein Dephosphorylation; Protein Kinase; Protein phosphatase; Proteins; RNA; RNA Binding; RNA-Directed DNA Polymerase; Reaction; Regulation; Reticulocytes; Reverse Transcription; Risk; Role; System; Viral; Viral Core Proteins; Viral Packaging; Viral Proteins; Viral Reverse Transcription; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; base; chronic liver disease; insight; novel; overexpression; pathogen; protein protein interaction; targeted agent; virus envelope

Abstract Hepatitis B virus (HBV) is a major cause of chronic viral hepatitis that increases dramatically the risk of liver cancer and other end-stage liver diseases such as cirrhosis. HBV is a small DNA virus and replicates its DNA genome via reverse transcription of a RNA intermediate called the pregenomic RNA (pgRNA). Viral replication depends critically on the assembly of a nucleocapsid (NC) that is composed of the viral core or capsids protein (HBc) and encapsidates a copy each of pgRNA and the reverse transcriptase (RT), which converts the RNA pregenome to the DNA genome within the NC. Viral capsids also encapsidate the host cyclin-dependent kinase 2 (CDK2) via unknown mechanisms. Challenging the current dogma that capsid assembly depends solely on the N-terminal domain (NTD) of HBc, we have recently discovered that under physiological conditions, capsid assembly critically depends on the C-terminal domain (CTD) of HBc. Furthermore, we have developed a mammalian cell-free system that recapitulates CTD-dependent capsid assembly and further regulates capsid assembly through host-mediated CTD phosphorylation and dephosphorylation. The cell-free capsid assembly system also recapitulates the specific encapsidation of CDK2. Building on these developments, we propose to dissect the role of CTD, and its state of phosphorylation as regulated by cellular protein kinases and phosphatases, in capsid assembly (Specific Aim 1). We also plan to elucidate the HBc and CDK2 requirements for CDK2 encapsidation (Specific Aim 2). Furthermore, we have recently developed methods to isolate the viral pgRNA in complex with RT, which is the substrate recognized by HBc during NC assembly to achieve specific encapsidation of pgRNA and RT. We now propose to combine the isolation of the pgRNA-RT complex together with the cell-free capsid assembly system to develop cell-free systems for the specific packaging of pgRNA (and RT) into NCs (Specific Aim 3).

摘要 B型肝炎病毒（HBV）是慢性病毒性肝炎的主要病因，其显著增加肝脏损害的风险。 癌症和其他终末期肝病如肝硬化。HBV是一种小DNA病毒， 通过称为前基因组RNA（pgRNA）的RNA中间体的逆转录，在基因组中表达。病毒复制 关键取决于由病毒核心或衣壳蛋白组成的核衣壳（NC）的组装 (HBc)并使pgRNA和逆转录酶（RT）各一个拷贝发生酶解，逆转录酶（RT）将RNA 前基因组到NC内的DNA基因组。病毒衣壳还使宿主细胞周期蛋白依赖性 激酶2（CDK 2）通过未知的机制。推翻了目前的教条，即衣壳组装取决于 仅仅在HBc的N-末端结构域（NTD）上，我们最近发现，在生理条件下， 在这种条件下，衣壳组装严重依赖于HBc的C-末端结构域（CTD）。此外，我们还 开发了一种哺乳动物无细胞系统，该系统重现CTD依赖性衣壳组装，并进一步 通过宿主介导的CTD磷酸化和去磷酸化调节衣壳组装。无细胞 衣壳组装系统也概括了CDK 2的特异性衣壳化。根据这些 发展，我们建议解剖CTD的作用，其磷酸化状态受细胞调节， 蛋白激酶和磷酸酶，在衣壳装配（具体目标1）。我们还计划阐明HBc， CDK 2抑制的CDK 2要求（具体目标2）。此外，我们最近开发了 分离与RT复合的病毒pgRNA的方法，RT是NC期间HBc识别的底物 组装以实现pgRNA和RT的特异性包裹。我们现在建议将pgRNA的分离与RT的特异性包裹结合起来。联合收割机 pgRNA-RT复合物与无细胞衣壳组装系统一起开发用于重组蛋白的无细胞系统。 将pgRNA（和RT）特异性包装到NC中（特异性目标3）。