RNA-Protein Interactions During Hepatitis C Virus Infection

丙型肝炎病毒感染期间的 RNA-蛋白质相互作用

• 批准号: 9317274
• 负责人: Rebecca Lynn Adams
• 金额: $ 5.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9317274
• 关键词: Affect; Alpha Cell; Antisense RNA; Antiviral Agents; Antiviral Therapy; Basic Science; Binding Sites; Cancer Etiology; Cell Culture Techniques; Cell physiology; Cells; Cessation of life; Chromatography; Chronic; Cirrhosis; Clip; Complex; Development; Elements; Gene Expression; Genome; Genotype; Goals; Hepatitis C; Hepatocyte; Immunoprecipitation; In Vitro; Infection; Investigation; Knowledge; Lead; Liver Cirrhosis; Malignant Neoplasms; Mass Spectrum Analysis; MicroRNAs; Modeling; Molecular; Mutation; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Play; Population; Primary carcinoma of the liver cells; Protease Domain; Proteins; RNA; RNA Binding; RNA Helicase; RNA Virus Infections; RNA Viruses; RNA analysis; RNA-Protein Interaction; Ribonucleosides; Role; Structure; Testing; Transcript; Translating; Vaccines; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; Work; base; cellular targeting; combat; crosslink; experimental study; follow-up; helicase; in vivo; knock-down; liver development; liver transplantation; metaplastic cell transformation; mortality; mutant; novel; prevent; research study; transcriptome sequencing; viral RNA; viral detection

Project Summary Hepatitis C Virus (HCV) is a RNA virus that currently infects three percent of the world's population. By chronically infecting hepatocytes, HCV leads to severe physiological changes of host cells. This often leads to liver cirrhosis, which is the leading cause for development of hepatocellular carcinoma (HCC) and need for liver transplantation. HCC is the second leading cause of cancer-related deaths worldwide, and current treatment of HCC is indirect and ineffective. Vaccines have not been successfully developed to prevent HCV infection, and direct-acting antiviral (DAA) therapy has only recently become successful, largely thanks to basic science research studying molecular aspects of HCV infection. HCV is a RNA-only virus, and recent advances in the study and analysis of RNA has lead to a revolution in understanding the complex role that diverse RNAs play in cells. This proposal seeks a molecular understanding of RNA-protein interactions between the virus and host cell during HCV infection using state-of- the-art approaches. In Aim 1, I will analyze the in vivo RNA targets of the HCV bi-functional protease/RNA helicase, NS3. Although NS3 is a target of current DAA therapy, no studies have analyzed what the RNA substrates for NS3 helicase activity are. It is presumed that NS3 targets viral RNA, but it is unknown whether the helicase impacts cellular gene expression. To resolve these unknowns, I will perform PAR-Clip on NS3. PAR-Clip permits unambiguous identification of regions of RNA that directly interact with proteins, and I will use a variety of mutants and/or drugs to probe the consequences of altering helicase or protease activity. In Aim 2, I will test whether the HCV RNA genome interacts with cellular proteins. The Pyle lab has uncovered conserved RNA structures that are required for normal HCV replication and infectivity. I will use RAP-MS to uncover whether host cell proteins interact with these structures. Follow-up experiments will confirm direct interactions and analyze the function of these interactions. These approaches will inform how RNA-protein interactions impact viral replication and cellular physiology. The ultimate goal of this work is to aid in the development of DAAs, uncover markers for HCC progression, and discover aspects of infection that can inform the study of other RNA viruses.

项目摘要 丙型肝炎病毒（HCV）是一种RNA病毒，目前感染了世界人口的三％。经过 长期感染肝细胞，HCV导致宿主细胞的严重生理变化。这通常会导致 肝肝硬化，这是肝细胞癌（HCC）发展的主要原因，需要 肝移植。 HCC是全球与癌症相关死亡的第二大原因，当前 HCC的治疗是间接和无效的。疫苗尚未成功开发以防止HCV 感染和直接作用抗病毒药（DAA）疗法直到最近才成功，这在很大程度上要归功于基本 科学研究研究HCV感染的分子方面。 HCV是一种仅RNA的病毒，RNA研究和分析的最新进展导致了一场革命 了解各种RNA在细胞中起的复杂作用。该建议寻求分子 使用最新的HCV感染期间病毒与宿主细胞之间的RNA-蛋白质相互作用的理解 艺术的方法。 在AIM 1中，我将分析HCV双官能蛋白酶/RNA解旋酶NS3的体内RNA靶标。虽然 NS3是当前DAA治疗的靶标，没有研究分析了NS3解旋酶的RNA底物 活动是。假定NS3靶向病毒RNA，但尚不清楚解旋酶是否影响细胞 基因表达。为了解决这些未知数，我将在NS3上执行par-clip。 PAR-CLIP允许明确 鉴定直接与蛋白质相互作用的RNA区域，我将使用多种突变体和/或 药物以探测改变解旋酶或蛋白酶活性的后果。 在AIM 2中，我将测试HCV RNA基因组是否与细胞蛋白相互作用。 Pyle Lab发现了 正常HCV复制和感染性所需的保守RNA结构。我将使用RAP-MS 发现宿主细胞蛋白是否与这些结构相互作用。后续实验将直接确认 相互作用并分析这些相互作用的功能。 这些方法将告知RNA-蛋白质相互作用如何影响病毒复制和细胞生理。 这项工作的最终目标是帮助发展DAA，发现HCC进展的标记以及 发现感染的各个方面，可以告知其他RNA病毒的研究。