Crystallizing Hepatitis B Virus Reverse Transcriptase

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。HBV是肝病毒组的一员，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。为了进行这种不寻常的复制周期，所有肝病毒都编码一种多功能逆转录酶（RT）。HBV复制的关键早期步骤是在病毒逆转录酶（RT）和位于pgRNA 5'端称为Epsilon的特定RNA信号之间组装特定核糖核蛋白（RNP）复合物。使用epsilon RNA作为模板，RT能够从头开始DNA合成（不需要任何核酸引物），使用自身作为蛋白质引物（蛋白质引物反应）。RT-epsilon复合体还作为一个包装信号，启动病毒核衣壳的组装，导致RT和pgRNA选择性地结合到逆转录区域。因此，RT的功能，特别是其与Epsilon RNA的相互作用及其蛋白引物活性，对HBV的组装和复制至关重要，因此是开发特异性抗HBV药物的绝佳靶点。我们最近在表达和纯化活性重组HBV RT蛋白方面的成功，带来了令人兴奋的可能性，现在可以纯化足够数量的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射线晶体学技术研究RT函数。充分量的RT蛋白和RT-epsilon复合物将被纯化用于结晶，长期目标是获得它们的高分辨率结构。此外，RT蛋白的一个新的结构域，称为TP结构域，将被纯化和结晶。为了实现这些目标，目前的细菌表达系统将扩大规模，如有必要，真核表达系统将被采用。与其他生化研究一起，本文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靶向RT-epsilon相互作用和蛋白启动的新型抗hbv药物，从而有助于预防hbv诱导的肝癌和其他致命疾病的发展。

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.