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.
