Crystallizing Hepatitis B Virus Reverse Transcriptase

结晶乙型肝炎病毒反转录酶

• 批准号: 7033057
• 负责人: Hwai-Chen Guo
• 金额: $ 23.66万
• 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 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033057
• 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）：丙型肝炎病毒（HBV）仍然是慢性肝炎，肝硬化和肝细胞癌的主要原因。 HBV是Hepadnavirus组的成员，双链DNA病毒。它通过新型的逆转录途径通过RNA中间体（前基因组RNA或PGRNA）复制。为了执行这个不寻常的复制周期，所有肝病毒病毒均编码多功能逆转录酶（RT）。 HBV复制中的关键早期步骤是病毒逆转录酶（RT）和称为Epsilon的特定RNA信号之间的特定核糖核蛋白（RNP）复合物的组装，位于PGRNA的5'端。将EPSILON RNA作为模板，RT能够将DNA合成de Noko（无需任何核酸引物）启动，将其本身用作蛋白质引物（蛋白质启动反应）。 RT-EPSILON复合物还充当一个包装信号，可以启动病毒核的组装，从而导致将RT和PGRNA选择性掺入逆转录区域。因此，尤其是RT功能，它与Epsilon RNA及其蛋白启动活性的相互作用对于HBV组装和复制至关重要，因此代表了开发特定抗HBV剂的出色靶标。我们最近在表达和净化活性的，重组HBV RT蛋白方面的成功带来了令人兴奋的可能性，即现在可以使用X射线晶体学来纯化足够量的RT蛋白来进行高分辨率的结构研究。因此，在此探索性项目中提出了应用X射线晶体学技术来研究RT功能的技术。 足够量的RT蛋白和RT-EPSILON复合物将被纯化以进行结晶，其长期目标是获得其高分辨率结构。另外，将纯化并结晶的RT蛋白的新型域（称为TP结构域）。为了实现这些目标，将对当前的细菌表达系统进行缩放，并在必要时进行真核表达系统。与其他生化研究一起，此处提出的高分辨率结构研究将大大增强我们对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促进针对RT- EPSILON相互作用和蛋白质启动的新型抗HBV药物的基于结构的合理设计，这反过来又有助于防止HBV诱导的肝癌和其他致命疾病的发展。