The Structural Biology of HBV

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Summary 240 million people suffer from chronic Hepatitis B Virus infection (HBV). In vivo, assembly of new virions begins with a complex of the viral polymerase with a stem loop on the viral RNA. This complex nucleates assembly of a T=4 capsid resulting in the RNA-filled core. Within the RNA-filled core the polymerase becomes active and reverse transcribes the linear single stranded RNA pregenome to the relaxed circular double strand DNA of mature HBV cores. Mature cores and empty cores, but not immature cores, display signals that allow their transport to the nucleus or the ER. These directions respectively maintain chronic infection or result in envelopment and secretion from the cell. Our preliminary studies show that viral RNA lines the interior surface of the capsid and supports the hypothesis that in RNA-filled cores the nucleating polymerase complex occupies a specific site (or a small number of sites) with respect to the capsid. We further propose that that the polymerase travels on the RNA during reverse transcription. We propose that owing to its stiffness, the dsDNA genome of mature capsids must adopt a completely different organization. We will investigate the structure and biochemistry of empty, RNA-filled, and mature DNA-filled capsid. Our overarching goals are to describe the mechanism of reverse transcription and the structural basis for signaling maturation of the HBV genome. Towards this end we will develop purification strategies for purifying different classes of core, examine their dynamic properties using biophysical techniques, use single molecule techniques to determine variability of core mass, and investigate the reverse transcription reaction itself. In a second set of aims we will determine structures of RNA-filled and DNA-filled cores to investigate the disposition of the nucleic acid, its effects on capsid symmetry, and the availability of capsid binding sites for host proteins, such as the importins responsible for nuclear import. Treatment of chronic HBV with reverse transcriptase inhibitors, the standard of care, decreases viral load and improves liver condition but it rarely leads to a “cure”, even after years of treatment. Remarkably, there is no real understanding of the structural basis of reverse transcription and how a signal for nucleic acid state is transduced to allow the virus lifecycle to progress. Addressing these deficits is the goal of this proposal.