Dissecting the molecular determinants of hepatitis B tropism

摘要 乙肝是一个全球性的健康问题，约有3.6亿至4亿慢性感染者。这些 个人可能会发展成纤维化、肝硬变和肝细胞癌，导致频繁死亡。一个 预防性疫苗是可用的，也有抗乙肝药物可以抑制病毒血症，但没有治愈方法。 乙肝的病原体是乙肝病毒，一种小的双链DNA病毒(3.2kb 基因组)有四个重叠的开放阅读框架(ORF‘s)，产生4个基因产物。缺乏一种 乙肝病毒的小动物模型阻碍了对乙肝病毒的研究和更有效的治疗方法的开发。 乙肝病毒的宿主范围很窄，只感染人类和黑猩猩。这一高度受限的基础 物种取向不被理解，这是我研究的重点。 以往的数据表明，当传染性的乙肝病毒粒子组装并从小鼠体内释放时 该物种不支持细胞、病毒进入和基因组复制。我的目标是系统地 剖析乙肝病毒生命周期中的障碍，重点放在啮齿动物细胞对乙肝病毒的吸收上。最近，人类的钠 牛磺胆酸共转运多肽(HNTCP)被认为是乙肝病毒和丁型肝炎病毒进入的受体。 HNTCP在小鼠细胞中的表达将促进丁型肝炎病毒(HDV)的摄取，HDV是一种卫星病毒，可 使用乙肝病毒包膜蛋白来产生感染性病毒粒子，但不是乙肝病毒。这表明，要么有 除了hNTCP以外的其他人类宿主因素是乙肝病毒进入所必需的，或者有小鼠占主导地位 限制乙肝病毒吸收的负面因素。 本研究的目的是了解乙肝病毒的限制性取向，并确定影响其功能的因素 是进入和理解它们的功能所必需的。拟议的研究将导致几个 取得了重大进展。首先，开发出一种新型的乙肝病毒报告系统，以 证明两个重要的原则，1)它们作为乙肝病毒进入的报告的实用性，2)表达的能力 乙肝病毒粒子内的异源蛋白质。其次，将进行基因筛查，以确定其他 除了hNTCP外，人类宿主因素也是乙肝病毒感染所必需的。总的来说，成功的 该项目的完成将产生新的工具来调查乙肝病毒的进入和人类身份的识别 乙肝病毒进入所需的宿主因素。新人类宿主的鉴定与鉴定 对乙肝病毒感染至关重要的因素将形成建立可遗传的小鼠模型的蓝图 对乙肝病毒感染易感性。该模型可用于新疗法的测试。

Summary Hepatitis B is a global health problem with ~360-400 million chronically infected patients. These individuals can develop fibrosis, cirrhosis and hepatocellular carcinoma resulting frequently in death. A prophylactic vaccine is available and there are anti-HBV drugs that can suppress viremia but there is no cure. The causative agent of hepatitis B is the hepatitis B virus (HBV), a small double stranded DNA virus (3.2 kb genome) with four overlapping open reading frames (ORF's), which produces 4 gene products. The lack of a small animal model for HBV has hampered the study of HBV and the development of more effective therapies. HBV has a narrow host range infecting only humans and chimpanzees. The basis for this highly restricted species tropism is not understood and is the focus of my studies. Previous data demonstrate that while infectious HBV virions assemble and are released from mouse cells, viral entry and genome replication are not supported in this species. It is my goal to systematically dissect blocks in the HBV life-cycle focusing here on HBV uptake in rodent cells. Recently, the human sodium taurocholate co-transporting polypeptide (hNTCP) has been identified as a receptor for HBV and HDV entry. Expression of hNTCP in murine cells will facilitate the uptake of hepatitis delta virus (HDV), a satellite virus that uses the HBV envelope proteins to produce infectious virions, but not HBV. This suggests that there are either additional human host factors besides hNTCP necessary for HBV entry or that there are murine dominant negative factors that restrict HBV uptake.  The purpose of this study is to understand the restrictive tropism of HBV and to identify factors that are necessary for entry and to understand their function. The proposed study will result in several significant advancements. First, the development of a novel HBV reporter systems will be created to demonstrate two important principles, 1) their utility as a reporter for HBV entry, and 2) the ability to express heterologous proteins within a HBV virion. Secondly, a genetic screen will be performed to identify additional human host factors besides hNTCP that are necessary for HBV uptake. Collectively, the successful completion of this project will result in novel tools to investigate HBV entry and the identification of human host factors necessary for HBV entry. The identification and characterization of additional human host factors that are important for HBV uptake will form the blue-print to create a mouse model with inheritable susceptibility to HBV infection. This model could be used for testing of novel therapies.