Structural & Functional Investigations of Hepatitis B Virus Pol Activity in a Native-like Context

B型肝炎病毒是世界范围内肝癌的主要原因。尽管有有效、安全和廉价的疫苗，但每年仍有大约100万人新感染。他们加入了一个约2.4亿患者的队列，这些患者在未能完全清除初始暴露于病毒后患有慢性HBV感染。这些人目前的前景很差，因为主要的临床抗病毒药物，针对病毒聚合酶的逆转录酶活性位点，不能根除病毒，因此意味着终身治疗。可悲的是，在这种治疗几十年后，HBV诱导的肝癌死亡率最多仅降低了4倍。每年大约有70多万人死于感染，导致肝衰竭、肝硬化和肝癌。世界卫生组织已经发布了一项全球挑战，到2030年使HBV感染成为可治疗的疾病。应对这一挑战的一个主要障碍是难以在其天然环境中研究病毒聚合酶-由病毒编码的核心蛋白组成的病毒诱导蛋白容器，其中病毒核酸从单链RNA形式转化为切口双链DNA形式，这是肝脏中持续感染的来源。这种困难主要是由于聚合酶，它在大多数条件下溶解性很差。我们已经开发出一种独特的系统，可以在大肠杆菌中安全地进行研究，其中聚合酶通过结合其天然RNA靶点，即天然病毒蛋白质外壳内的茎环来稳定。我们将使用这个系统来研究酶如何在这个专门的蛋白质容器内实现这些潜在的致命核酸转化，使用光谱技术来测定酶促反应。这些将与我们世界一流的电子显微镜实验室的最先进的结构研究相结合，以及我们为研究病毒内核酸结构而开发的专业技术。这些将使我们能够探索领先的新型抗病毒候选药物的作用机制，并使该系统广泛用于快速治疗开发。

Hepatitis B Virus is the major cause of liver cancer worldwide. Despite the availability of an effective, safe and cheap vaccine roughly a million people are newly infected each year. They join a cohort of about 240 million patients who suffer from chronic HBV infection after failing to fully clear an initial exposure to the virus. The current outlook for these people is poor since the major clinical anti-viral drugs, directed at the reverse transcriptase active site of the viral polymerase, do not eradicate virus, and therefore imply lifelong treatment. Sadly, death from HBV induced liver cancer is lowered only by a maximum of 4-fold after decades of this treatment. Over 700,000 people die roughly every year as a result of infection which causes liver failure, cirrhosis and liver cancer. The WHO have issued a Global Challenge to make HBV infection a treatable disease by 2030. A major barrier to meeting this challenge is the difficulty in studying the viral polymerase in its native environment - a virally-induced protein container composed of virally-encoded core protein, in which the viral nucleic acid is converted from a single-stranded RNA form to a nicked double-stranded DNA version, the source of continued infection in the liver. This difficulty is principally due to the polymerase, which is only poorly soluble in most conditions. We have developed a unique system that can be studied safely in the bacterium E.coli, in which the polymerase is stabilised by binding to its native RNA target, the epsilon stem-loop, within a native-like viral protein shell. We will use this system to investigate how the enzyme achieves these potentially lethal nucleic acid transformations within this specialised protein container using spectroscopic techniques to assay the enzymatic reactions. These will be coupled with state-of-the-art structural studies in our world-class electron microscopy laboratory, and a specialised technique we have developed for studying the structures of nucleic acids within viruses. These will allow us to explore the mechanisms of action of leading novel anti-viral drug candidates, and make this system widely available for rapid therapeutic exploitation.