The Structural Biology of HBV

摘要 2.4亿人患有慢性乙肝病毒感染(乙肝病毒)。在体内，组装新的 病毒粒子开始于病毒聚合酶与病毒RNA上的茎环的复合体。这个建筑群 使T=4的衣壳组装成核，从而形成充满RNA的核心。在充满RNA的核心内 聚合酶变得活跃，并将线性单链RNA前基因组反向转录到 松弛的成熟乙肝病毒核心的环状双链DNA。成熟核和空核，但不是 未成熟的核心，显示信号，允许它们传输到细胞核或内质网。这些方向 分别维持慢性感染或导致细胞包膜和分泌。我们的 初步研究表明，病毒RNA排列在衣壳的内表面，支持 假设在充满RNA的核心中，成核聚合酶复合体占据特定的位置(或 少数地点)相对于衣壳。我们进一步提出，聚合酶可以通过 在反转录过程中的RNA上。我们认为，由于它的僵硬，dsDNA基因组 成熟的衣帽必须采用一个完全不同的组织。我们将调查其结构和 空的、充满RNA的和成熟的充满DNA的衣壳的生物化学。我们的首要目标是 描述逆转录的机制和信号成熟的结构基础 乙肝病毒基因组。为此，我们将制定净化策略，以净化不同的 利用生物物理技术检查其动力学性质的岩心类别，使用单分子 确定核心质量的可变性和研究逆转录反应的技术 它本身。在第二组目标中，我们将确定填充RNA和DNA的核心的结构，以 研究核酸的分布，它对衣壳对称性的影响，以及病毒的可用性 宿主蛋白的衣壳结合部位，例如负责核进口的重要蛋白。 用逆转录酶抑制剂治疗慢性乙肝，这是治疗的标准，可减少病毒 但即使在多年的治疗后，它也很少能“治愈”。 值得注意的是，没有真正了解逆转录的结构基础，以及如何 核酸状态的信号被转导，以允许病毒生命周期的进展。解决这些问题 赤字是这项提案的目标。

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.