Crystallizing Hepatitis B Virus Reverse Transcriptase

结晶乙型肝炎病毒反转录酶

• 批准号: 6851919
• 负责人: Hwai-Chen Guo
• 金额: $ 20.19万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6851919
• 关键词: RNA directed DNA polymerase; SDS polyacrylamide gel electrophoresis; X ray crystallography; affinity chromatography; crystallization; hepatitis B virus group; protein protein interaction; protein purification; protein structure function; recombinant proteins; ribonucleoproteins; structural biology; tissue /cell culture; transcription factor; transfection /expression vector; virus protein; virus replication

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) remains a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HBV is a member of the hepadnavirus group, double-stranded DNA viruses. It replicates through an RNA intermediate (the pregenomic RNA, or pgRNA) by a novel reverse transcription pathway. To carry out this unusual replication cycle, all hepadnaviruses encode a multi-functional reverse transcriptase (RT). A critical early step in HBV replication is the assembly of a specific ribonucleoprotein (RNP) complex between the viral reverse transcriptase (RT) and a specific RNA signal called Epsilon, located at the 5' end of the pgRNA. Using epsilon RNA as a template, the RT is able to initiate DNA synthesis de novo (without the need of any nucleic acid primers), using itself as a protein primer (the protein priming reaction). The RT-epsilon complex also acts as a packaging signal to initiate assembly of the viral nucleocapsids, leading to the selective incorporation of both the RT and the pgRNA into the locale of reverse transcription. Therefore, RT functions, in particular, its interaction with the Epsilon RNA and its protein priming activity, are critical for HBV assembly and replication and as such, represent excellent targets to develop specific anti-HBV agents. Our recent success in expressing and purifying active, recombinant HBV RT proteins has brought about the exciting possibility that sufficient amounts of RT proteins could now be purified for high-resolution structural studies using X-ray crystallography. Therefore, it is proposed in this exploratory project to apply the technology of X-ray crystallography to study the RT functions. Sufficient amounts of RT proteins, and the RT-epsilon complex will be purified for crystallization, with the long-term objective of obtaining their high-resolution structures. In addition, a novel domain of the RT protein, called TP domain, will be purified and crystallized. To accomplish these goals, the current bacterial expression system will be scaled up and if necessary, eukaryotic expression systems will be adopted. Together with other biochemical studies, the high-resolution structure studies proposed here will greatly enhance our understanding of the mechanisms of RT-epsilon interaction and protein priming. Furthermore, the anticipated results from biochemical and structural studies will facilitate structure-based rational design of novel anti-HBV agents targeting at RT-epsilon interaction and protein priming, which, in turn, will help to prevent the development of HBV-induced liver cancer and other fatal diseases.

描述（由申请人提供）：乙型肝炎病毒（HBV）仍然是慢性肝炎、肝硬化和肝细胞癌的主要原因。乙型肝炎病毒是嗜肝DNA病毒组的成员，双链DNA病毒。它通过一种新的逆转录途径通过 RNA 中间体（前基因组 RNA，或 pgRNA）进行复制。为了执行这种不寻常的复制周期，所有嗜肝DNA病毒都编码一种多功能逆转录酶（RT）。 HBV 复制的一个关键的早期步骤是在病毒逆转录酶 (RT) 和位于 pgRNA 5' 端的称为 Epsilon 的特定 RNA 信号之间组装特定的核糖核蛋白 (RNP) 复合物。使用 epsilon RNA 作为模板，RT 能够使用自身作为蛋白质引物（蛋白质引发反应），从头开始 DNA 合成（无需任何核酸引物）。 RT-epsilon 复合物还充当包装信号来启动病毒核衣壳的组装，从而导致 RT 和 pgRNA 选择性掺入逆转录区域。因此，RT 功能，特别是其与 Epsilon RNA 的相互作用及其蛋白质启动活性，对于 HBV 组装和复制至关重要，因此代表了开发特异性抗 HBV 药物的极好靶点。我们最近在表达和纯化活性重组 HBV RT 蛋白方面取得的成功带来了令人兴奋的可能性，即现在可以纯化足够量的 RT 蛋白，用于使用 X 射线晶体学进行高分辨率结构研究。因此，本探索性项目提出应用X射线晶体学技术来研究RT函数。 足够量的 RT 蛋白和 RT-ε 复合物将被纯化以进行结晶，长期目标是获得其高分辨率结构。此外，RT 蛋白的一个新结构域（称为 TP 结构域）将被纯化和结晶。为了实现这些目标，现有的细菌表达系统将被扩大规模，如有必要，将采用真核表达系统。与其他生化研究一起，这里提出的高分辨率结构研究将极大地增强我们对 RT-epsilon 相互作用和蛋白质启动机制的理解。此外，生化和结构研究的预期结果将有助于基于结构的合理设计针对RT-ε相互作用和蛋白质启动的新型抗HBV药物，这反过来又有助于预防HBV诱发的肝癌和其他致命疾病的发展。