'IMMATURE SECRETION' VARIANTS OF HUMAN HEPATITIS B VIRUS

我们的长期目标是了解病毒性肝炎的病理生物学。乙型肝炎病毒（HBV）是全球第四大最常见的人类感染源。慢性HBV感染与肝癌的发生密切相关。HBV慢性感染的建立机制及其发病机制仍有待充分探讨。最近，越来越多的证据表明HBV的慢性性可能受到随着肝病进展而在患者体内进化的遗传变异的影响。自聚合酶链反应技术发明以来，已经报道了许多HBV序列变异。然而，在HBV变异研究中一个常见的挫折是，大多数报道的序列变异没有已知的功能意义，也没有表现出与野生型病毒明显不同的强烈表型。HBV核心抗原中最常见的错义突变发生在全球慢性携带者（83%的肝癌患者）的密码子97处。我们最近对该突变体的功能表征揭示了一种新的强表型，称为“未成熟分泌”表型。与野生型HBV不同，密码子97突变体分泌的Dane颗粒主要含有HBV DNA基因组的未成熟形式（低分子量）。在本申请中，我们提出1)将我们对“未成熟分泌物”现象的观察从组织培养扩展到患者和动物模型。我们还试图将“未成熟分泌物”表型扩展到不同地理位置流行的其他病毒亚型。2)从遗传学和生物化学两方面探讨“未成熟分泌”的机制。需要测试的几个假设之一是，“未成熟分泌”可能是由野生型包膜和突变核心蛋白之间的超高效相互作用引起的。3)最后，我们将通过体外感染实验比较高频率密码子97突变体和野生型HBV的感染性。这项工作的成功完成将有助于了解HBV形态发生和病毒粒子分泌的基本规则，以及对慢性病毒性肝炎和许多其他慢性进行性病毒性疾病的重要意义。

Our long-term goal is to understand the pathobiology of viral hepatitis. Hepatitis B virus (HBV) is the 4th most common infectious agent in humans worldwide. Chronic infection with HBV is tightly associated with the development of liver cancer. The mechanism of how HBV chronic infection is established and its pathogenesis remains to be fully explored. Recently, there has been increasing evidence that HBV chronicity may be influenced by the presence of genetic variants that evolve in patients as liver disease progresses. Since the invention of polymerase chain reaction technology, many HBV sequence variants have been reported. However, a common frustration in the research of HBV variants is that the majority of these reported sequence variants has no known functional significance and does not exhibit a strong phenotype distinctively different from the wild type virus. The most frequent missense mutation found in the HBV core antigen occurs at codon 97 in chronic carriers worldwide (in 83 percent hepatoma patients). Our recent functional characterization of this mutant uncovered a novel and strong phenotype, dubbed an "immature secretion" phenotype. Unlike wild type HBV, the secreted Dane particles of codon 97 mutants contain predominantly the immature form (lower molecular weight) of the HBV DNA genome. In this application, we propose 1) to extend our observation of the "immature secretion" phenomenon from tissue culture to patients and animal models. We also attempt to extend the "immature secretion" phenotype to other viral subtypes prevalent in different geographic locations. 2) to investigate the mechanism of "immature secretion" by both genetic and biochemical approaches. One of the several hypotheses to be tested is that "immature secretion" could be caused by hyper-efficient interactions between wild type envelope and mutant core proteins. 3) Finally, we will compare the infectivity of the highly frequent codon 97 mutants and wild type HBV via in vitro infection assays. Successful completion of this work will help understand the fundamental rules governing HBV morphogenesis and virion secretion, in addition to the important implications for chronic viral hepatitis as well as many other chronic progressive viral diseases.