Transgenic mouse models of HCV infection and replication

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是慢性肝炎、肝硬变和肝细胞癌的主要原因。丙型肝炎病毒在美国慢性感染约400万人，在全球感染1.7亿人。丙型肝炎病毒与艾滋病毒的混合感染非常普遍，总共有25%-30%的艾滋病毒阳性者，特别是在吸毒者中，高达70%的混合感染[2]。丙型肝炎病毒感染是肝细胞癌发生的主要危险因素。丙型肝炎病毒相关的终末期肝病是肝移植的主要适应症。由于缺乏强大的细胞培养、丙型肝炎病毒繁殖系统和可靠的丙型肝炎病毒感染和复制的小动物模型，丙型肝炎病毒的研究进展受到了严重的阻碍。最近的突破是发展了用于复制丙型肝炎病毒复制子RNA和生产传染性丙型肝炎病毒的强大的细胞培养系统，这使得对整个丙型肝炎病毒生命周期的基因研究成为可能。然而，发展丙型肝炎病毒感染和复制的小动物模型是具有挑战性的，缺乏可靠的小动物模型是丙型肝炎研究的主要障碍。最近，我们已经证明，2a型丙型肝炎病毒RNA在小鼠胚胎成纤维细胞(MEF)中有效复制，但在小鼠肝细胞中复制效率较低。更重要的是，我们已经证明了细胞内的cDNA源的丙型肝炎病毒RNA导致了感染性丙型肝炎病毒的强劲产生。查理赖斯的研究小组最近的一项研究表明，人CD81和occludin在小鼠肝细胞中的表达是感染丙型肝炎病毒假型颗粒的必要条件和充分条件。这些显着的进展为开发新型的丙型肝炎病毒感染和复制转基因小鼠模型提供了坚实的基础。我们假设细胞类型特异的细胞蛋白对于有效的丙型肝炎病毒RNA复制是重要的，并且在肝细胞中表达人CD81和occludin的转基因小鼠容易感染丙型肝炎病毒。这一探索性研究方案的总体目标是开发新的转基因丙型肝炎病毒小鼠模型。具体地说，我们将1)利用蛋白质组学、生化和遗传互补方法(特异性目标1)确定对小鼠细胞中高效复制丙型肝炎病毒RNA至关重要的细胞蛋白；2)开发新型转基因小鼠，这些转基因小鼠要么包含表达荧光素酶的JFH1丙型肝炎病毒RNA的全长cDNA，要么在肝脏特异性启动子的控制下同时表达人CD81和occludin(特异性目标2)；以及3)使用表达荧光素酶的丙型肝炎病毒结合小鼠干扰素以及丙型肝炎病毒NS3蛋白酶和NS5B聚合酶特异性抑制物(特异性目标3)来确定转基因小鼠中的丙型肝炎病毒感染和复制。转基因丙型肝炎病毒小鼠模型的成功建立将代表着丙型肝炎病毒研究领域的范式转变，将使我们能够确定病毒和细胞蛋白在丙型肝炎病毒感染、复制、致病和癌变中的作用和作用机制，以及非法药物和酒精对丙型肝炎病毒复制和致病机制的影响。此外，转基因丙型肝炎病毒小鼠模型将有助于抗丙型肝炎病毒药物的发现和丙型肝炎病毒疫苗的开发，最终控制丙型肝炎病毒感染。 公共卫生相关性：丙型肝炎病毒(丙型肝炎病毒)感染仍然是一个主要的全球健康问题，影响着全球约1.7亿人和400万美国人。我们这项研究的总体目标是建立新型的转基因丙型肝炎病毒小鼠模型，以确定丙型肝炎病毒复制、致病、致癌的分子机制，以及非法药物和酒精对丙型肝炎病毒复制和发病机制的影响。此外，转基因的丙型肝炎病毒小鼠模型将有助于抗病毒药物的发现和疫苗的开发，最终控制丙型肝炎病毒感染。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HCV chronically infects approximately 4 million people in the U.S. and 170 million people worldwide. HCV co-infection with HIV is very common with overall 25-30% of HIV-positive persons, particularly among drug abusers with up to 70% co-infection (2). HCV infection is a major risk factor for HCC development. HCV- associated end-stage of liver diseases is the leading indication for liver transplantation. Advances on HCV research have been significantly hampered by the lack of a robust cell culture HCV propagation system and reliable small animal models of HCV infection and replication. Recent breakthroughs have been the development of robust cell culture systems for replication of HCV replicon RNAs and production of infectious HCV, which allows genetic studies of the entire HCV life cycle. However, development of small animal models of HCV infection and replication is challenging and that the lack of a reliable small animal model represents a major barrier to HCV research. Recently, we have demonstrated that genotype 2a HCV RNA replicated efficiently in mouse embryonic fibroblasts (MEF) albeit inefficiently in mouse hepatocytes. More importantly, we have demonstrated that the cDNA-derived HCV RNA inside the cell resulted in robust production of infectious HCV. A recent study by Charlie Rice's group has demonstrated that the expression of human CD81 and occludin in mouse hepatocytes is essential and sufficient for infection of HCV pseudotyped particles. These remarkable advances provide a firm foundation to develop novel transgenic mouse models of HCV infection and replication. We hypothesize that cell type-specific cellular proteins are important for efficient HCV RNA replication and that transgenic mice expressing human CD81 and occludin in hepatocytes are susceptible to HCV infection. The overall goal of this exploratory research proposal is to develop novel transgenic HCV mouse models. Specifically, we will 1) identify cellular proteins important for efficient HCV RNA replication in mouse cells using proteomics, biochemical, and genetic complementation approaches (Specific Aim 1); 2) develop novel transgenic mice that either contain full-length cDNAs of luciferase-expressing JFH1 HCV RNAs or express both human CD81 and occludin under the control of a liver-specific promoter (Specific Aim 2); and 3) determine HCV infection and replication in transgenic mice using luciferase-expressing HCV in conjunction with the administration of mouse interferon and HCV NS3 protease- and NS5B polymerase-specific inhibitors (Specific Aim 3). The successful development of transgenic HCV mouse models will represent a paradigm shift in the field of HCV research and will allow us to determine the roles and mechanisms of action of viral and cellular proteins in HCV infection, replication, pathogenesis, and carcinogenesis as well as the effects of illicit drugs and alcohol on HCV replication and pathogenesis. Additionally, transgenic HCV mouse models will facilitate the anti-HCV drug discovery and HCV vaccine development to eventually control HCV infection. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infection remains a major global health problem, affecting approximately 170 million people worldwide and 4 million Americans. Our overall goal of this research proposal is to develop novel transgenic HCV mouse models for the determination of the molecular mechanisms of HCV replication, pathogenesis, carcinogenesis, and the effects of illicit drugs and alcohol on HCV replication and pathogenesis. Additionally, transgenic HCV mouse models will facilitate the antiviral drug discovery and vaccine development to eventually control HCV infection.