Transgenic mouse models of HCV infection and replication

描述（由申请人提供）：丙型肝炎病毒（HCV）是慢性肝炎、肝硬化和肝细胞癌（HCC）的主要原因。HCV慢性感染在美国约有400万人，全球约有1.7亿人。丙型肝炎病毒与艾滋病毒合并感染非常常见，在艾滋病毒阳性者中占25% -30%，特别是在吸毒者中，合并感染高达70%(2)。HCV感染是HCC发展的主要危险因素。HCV相关的终末期肝病是肝移植的主要指征。由于缺乏强大的细胞培养HCV繁殖系统和可靠的HCV感染和复制小动物模型，HCV研究的进展受到严重阻碍。最近的突破是开发了用于复制HCV复制子rna和生产感染性HCV的强大细胞培养系统，这使得对HCV整个生命周期的遗传研究成为可能。然而，HCV感染和复制的小动物模型的开发具有挑战性，缺乏可靠的小动物模型是HCV研究的主要障碍。最近，我们已经证明基因2a型HCV RNA在小鼠胚胎成纤维细胞（MEF）中高效复制，但在小鼠肝细胞中低效复制。更重要的是，我们已经证明了细胞内dna衍生的HCV RNA导致感染性HCV的强劲生产。Charlie Rice小组最近的一项研究表明，人类CD81和occludin在小鼠肝细胞中的表达对于HCV假型颗粒的感染是必要和充分的。这些显著的进展为开发新的HCV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性细胞蛋白对HCV RNA的高效复制很重要，并且在肝细胞中表达人CD81和occludin的转基因小鼠易受HCV感染。本探索性研究计划的总体目标是开发新型转基因HCV小鼠模型。具体来说，我们将1)利用蛋白质组学、生化和遗传互补方法鉴定对小鼠细胞中高效复制HCV RNA重要的细胞蛋白（Specific Aim 1）；2)在肝脏特异性启动子的控制下，开发含有表达荧光素酶的JFH1 HCV rna的全长cdna或同时表达人CD81和occludin的新型转基因小鼠（Specific Aim 2）；3)利用表达荧光素酶的HCV结合小鼠干扰素和HCV NS3蛋白酶和NS5B聚合酶特异性抑制剂，检测转基因小鼠的HCV感染和复制（Specific Aim 3）。转基因HCV小鼠模型的成功开发将代表HCV研究领域的范式转变，并将使我们能够确定病毒和细胞蛋白在HCV感染、复制、发病机制和癌变中的作用和作用机制，以及非法药物和酒精对HCV复制和发病机制的影响。此外，转基因HCV小鼠模型将促进抗HCV药物的发现和HCV疫苗的开发，最终控制HCV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.