Dissecting the molecular determinants of hepatitis B tropism

剖析乙型肝炎嗜性的分子决定因素

• 批准号: 9189982
• 负责人: Benjamin Y. Winer
• 金额: $ 4.36万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189982
• 关键词: Animal Model; Applications Grants; Candidate Disease Gene; Cells; Cellular Tropism; Cessation of life; Chronic; Cirrhosis; Clone Cells; Data; Data Reporting; Development; Dominant-Negative Mutation; Double Stranded DNA Virus; Engineering; Fibrosis; Genetic Screening; Genome; Goals; HBV Animal Model; Hepatitis B; Hepatitis B Transmission; Hepatitis B Virus; Hepatitis C virus; Hepatitis Delta Virus; Hepatocyte; Human; Individual; Infection; Infectious hepatitides; Integration Host Factors; Knock-out; Life Cycle Stages; Light; Modeling; Molecular; Monitor; Mus; Nature; Open Reading Frames; Pan Genus; Patients; Pharmaceutical Preparations; Plasmids; Polymerase; Predisposition; Primary carcinoma of the liver cells; Printing; Proteins; Reading Frames; Recombinants; Reporter; Rodent; Role; Satellite Viruses; Supporting Cell; System; Taurocholate Sodium; Testing; Tropism; Vaccines; Viral; Viral Genome; Viremia; Virion; Virus; Virus Diseases; Work; anti-hepatitis B; base; cDNA Library; curative treatments; effective therapy; env Gene Products; gene product; global health; hepatoma cell; heterokaryon; humanized mouse; in vivo; insight; intein; loss of function; mouse model; novel; novel therapeutics; polypeptide; prophylactic; receptor; research study; tool; uptake; virus development; virus envelope; virus tropism

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.

概括 乙型肝炎是一个全球性健康问题，约有 360-4 亿慢性感染患者。这些 个体可能会出现纤维化、肝硬化和肝细胞癌，常常导致死亡。一个 可以使用预防性疫苗，并且有抗乙型肝炎药物可以抑制病毒血症，但无法治愈。 乙型肝炎的病原体是乙型肝炎病毒 (HBV)，一种小型双链 DNA 病毒 (3.2 kb 基因组）具有四个重叠的开放阅读框（ORF），产生 4 个基因产物。缺乏一个 乙肝病毒的小动物模型阻碍了乙肝病毒的研究和更有效疗法的开发。 乙型肝炎病毒的宿主范围较窄，仅感染人类和黑猩猩。这种严格限制的基础 物种向性尚不为人所知，但它是我研究的重点。 先前的数据表明，虽然传染性 HBV 病毒粒子在小鼠体内组装并释放， 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 HBV 生命周期的各个部分，重点关注啮齿动物细胞中 HBV 的摄取。最近，人体钠 牛磺胆酸共转运多肽 (hNTCP) 已被确定为 HBV 和 HDV 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，这是一种卫星病毒， 使用 HBV 包膜蛋白产生感染性病毒粒子，但不产生 HBV。这表明有 除了 hNTCP 之外，还有其他人类宿主因素是 HBV 进入所必需的，或者存在鼠类显​​性因素 限制乙型肝炎病毒摄取的负面因素。  本研究的目的是了解 HBV 的限制性倾向并确定影响 HBV 的因素 是进入和理解其功能所必需的。拟议的研究将产生几个结果 重大进步。首先，将开发一种新型 HBV 报告系统 证明了两个重要原则，1）它们作为 HBV 进入报告者的效用，以及 2）表达的能力 HBV 病毒颗粒内的异源蛋白。其次，将进行基因筛查以确定额外的 除 hNTCP 外，人类宿主因素也是 HBV 摄取所必需的。总的来说，成功的 该项目的完成将产生新的工具来调查乙型肝炎病毒的进入和识别人类 乙肝病毒进入所必需的宿主因素。其他人类宿主的鉴定和表征 对 HBV 摄取重要的因素将构成创建具有可遗传性的小鼠模型的蓝图 乙肝病毒感染易感性。该模型可用于测试新疗法。