Transgenic mouse models of HCV infection and replication

HCV感染和复制的转基因小鼠模型

• 批准号: 8204439
• 负责人: Jieyun Jiang
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-03 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204439
• 关键词: Affect; Albumins; Alcohols; American; Animal Model; Antiviral Agents; Biochemical Genetics; CD81 gene; Catalytic RNA; Cell Culture System; Cell Culture Techniques; Cell Line; Cells; Chronic Hepatitis; Cirrhosis; Complementary DNA; DNA Polymerase II; Development; Embryo; Fibroblasts; Foundations; Genetic; Genetic Transcription; Genome; Genomics; Genotype; Goals; HCV Vaccine; HIV; HIV Seropositivity; Hepatitis C; Hepatitis C virus; Hepatitis Delta Virus; Hepatocyte; Human; Illicit Drugs; Infection; Infectious hepatitides; Interferons; Knowledge; Laboratories; Length; Life Cycle Stages; Liver; Liver diseases; Luciferases; Measures; Molecular; Mouse Cell Line; Mus; Pathogenesis; Peptide Hydrolases; Persons; Polymerase; Primary carcinoma of the liver cells; Production; Proteins; Proteomics; RNA; RNA Sequences; RNA replication; Replicon; Research; Research Proposals; Rice; Risk Factors; Role; Staging; Structure; System; Transgenic Mice; Transgenic Organisms; Variant; Viral; Viral Proteins; Virion; Virus; Virus Receptors; Virus Replication; Work; anti-hepatitis C; base; carcinogenesis; cell type; drug abuser; drug development; drug discovery; global health; hepatitis C virus NS3 protein; hepatoma cell; in vivo; inhibitor/antagonist; innovation; interdisciplinary approach; liver transplantation; mouse model; novel; occludin; particle; promoter; public health relevance; receptor; vaccine development; viral RNA

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.

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 长期感染美国约 400 万人和全球 1.7 亿人。 HCV 与 HIV 合并感染非常常见，占 HIV 阳性者总数的 25-30%，特别是在吸毒者中，合并感染率高达 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适应症。由于缺乏强大的细胞培养 HCV 传播系统和可靠的 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强大的细胞培养系统，用于 HCV 复制子 RNA 的复制和感染性 HCV 的生产，从而可以对整个 HCV 生命周期进行遗传学研究。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，我们证明基因型 2a HCV RNA 在小鼠胚胎成纤维细胞 (MEF) 中有效复制，尽管在小鼠肝细胞中复制效率较低。更重要的是，我们已经证明细胞内的 cDNA 衍生的 HCV RNA 导致感染性 HCV 的大量产生。 Charlie Rice团队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且充分的。这些显着的进展为开发新型 HCV 感染和复制转基因小鼠模型奠定了坚实的基础。我们假设细胞类型特异性细胞蛋白对于 HCV RNA 的有效复制很重要，并且在肝细胞中表达人 CD81 和 occludin 的转基因小鼠容易受到 HCV 感染。这项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) 使用蛋白质组学、生化和遗传互补方法鉴定对小鼠细胞中 HCV RNA 有效复制至关重要的细胞蛋白（具体目标 1）； 2) 开发新型转基因小鼠，其要么含有表达荧光素酶的 JFH1 HCV RNA 的全长 cDNA，要么在肝脏特异性启动子的控制下表达人 CD81 和 occludin（具体目标 2）； 3) 使用表达荧光素酶的 HCV 结合施用小鼠干扰素和 HCV NS3 蛋白酶和 NS5B 聚合酶特异性抑制剂来确定转基因小鼠中的 HCV 感染和复制（具体目标 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、发病机制和致癌作用中的作用和机制，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HCV小鼠模型将促进抗HCV药物的发现和HCV疫苗的开发，以最终控制HCV感染。 公共卫生相关性：丙型肝炎病毒 (HCV) 感染仍然是一个主要的全球健康问题，影响着全世界约 1.7 亿人和 400 万美国人。我们这项研究计划的总体目标是开发新型转基因 HCV 小鼠模型，用于确定 HCV 复制、发病机制、致癌作用的分子机制，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小鼠模型将促进抗病毒药物的发现和疫苗的开发，以最终控制HCV感染。

项目成果

Recent advance in antiviral drugs for hepatitis C.

• DOI: 10.3969/j.issn.1672-7347.2011.11.001
• 发表时间: 2011-11-01
• 期刊: Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
• 作者: Liu, Jia;Shi, Shuang;Luo, Guangxiang
• 通讯作者: Luo, Guangxiang