MODEL SYSTEMS FOR ANALYSIS OF HBV REPLICATION

用于分析 HBV 复制的模型系统

• 批准号: 2095253
• 负责人: Robert E LANFORD
• 金额: $ 23.19万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-05 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2095253
• 关键词: Baculoviridae; DNA directed RNA polymerase; RNA binding protein; RNA directed DNA polymerase; antiserum; disease /disorder model; epitope mapping; gene expression; hepatitis B antigens; hepatitis B virus group; laboratory rabbit; molecular cloning; nucleocapsid; oligonucleotides; site directed mutagenesis; tissue /cell culture; transposon /insertion element; virus genetics; virus protein; virus replication

Hepatitis B virus (HBV) represents a serious worldwide health problem. The acute infection is usually self-limiting but may prove fatal, and, in addition, up to 10% of infected individuals become chronic carriers of the disease. The total number of carriers has been estimated at 280 million. Chronicity frequently progresses to cirrhosis the liver and hepatocellular carcinoma. Chronic carriers represent the main reservoir of the virus, with transmission occurring through contaminated blood products, IV drug abuse, sexual contact, and from mother to infant at birth. A precise understanding of the mechanism of viral replication is required to devise strategies for eliminat-ing the carrier state. This proposal will address several unresolved issues pertaining to the replication of the viral genome and viral morphogenesis. The first specific aim is to examine core protein mutants for assembly and for encapsidation of pregenomic RNA, polymerase, and polymerase-RNA complexes. The proteins will be expressed in insect cells using a baculovirus expression system or in in vitro translation systems and in vitro assays will be developed to examine these functions. The ability of core mutants to complement the replication of a core minus HBV mutant will be examined in the human hepatocellular carcinoma cell line, HepG2. The mechanism of encapsidation and the functional significance of the virion associated kinase will be explored. The second specific aim will be to express polymerase and polymerase functional domains in insect cells and to develop in vitro assays for reverse transcription, pregenomic RNA binding, nucleotide binding, and RNase H activity. The third specific aim will be to examine the RNA sequence requirement for encapsidation of pregenomic RNA by core and for interaction of pregenomic RNA with polymerase. The in vitro RNA transcripts used in assays for core and polymerase functions will be altered to determine the minimal sequences required for recognition. The final specific aim will be to utilize the information obtained in the in vitro assays to make specific mutations in the HBV genome and examine replication the mutants in HepG2 cells.

丙型肝炎病毒（HBV）代表了严重的全球健康问题。这 急性感染通常是自限制的，但可能证明是致命的，并且在 此外，多达10％的感染者成为慢性载体 疾病。载体总数估计为2.8亿。 慢性性经常发展为肝硬化 癌。慢性载体代表病毒的主要储藏 通过受污染的血液制品，IV滥用药物进行的传播， 性接触，出生时从母亲到婴儿。精确的理解 需要病毒复制机制才能制定策略 消除载体状态。该建议将解决几个 与病毒基因组复制有关的未解决的问题和 病毒形态发生。第一个具体目的是检查核心蛋白 用于组装和封装基因组RNA，聚合酶的突变体， 和聚合酶-RNA复合物。蛋白质将以昆虫表示 使用杆状病毒表达系统或体外翻译的细胞 系统和体外测定法将开发以检查这些功能。 核心突变体补充核心负复制的能力 将在人肝细胞癌细胞中检查HBV突变体 线，hepg2。封装机制和功能意义 将探索与病毒与病毒相关的激酶的相关激酶。第二个特定目标 将在昆虫中表达聚合酶和聚合酶功能域 细胞并开发用于逆转录的体外测定，前基因组学 RNA结合，核苷酸结合和RNase H活性。第三个特定 目的是检查封装的RNA序列要求 通过核心和基因组RNA的相互作用的基因组RNA与 聚合酶。用于核心和 聚合酶功能将改变以确定最小序列 识别所需的。最终的具体目的是利用 在体外测定中获得的信息，以在 HBV基因组并检查HEPG2细胞中的突变体。