RNA-Protein Interactions During Hepatitis C Virus Infection

项目摘要 丙型肝炎病毒（HCV）是一种RNA病毒，目前感染世界人口的3%。通过 慢性感染肝细胞，HCV导致宿主细胞的严重生理变化。这往往导致 肝硬化是肝细胞癌（HCC）发展的主要原因， 肝移植HCC是全世界癌症相关死亡的第二大原因， HCC的治疗是间接的和无效的。预防HCV的疫苗尚未成功开发 直接作用的抗病毒（DAA）治疗直到最近才取得成功，这主要归功于基本的 研究HCV感染分子方面的科学研究。 HCV是一种仅有RNA的病毒，RNA研究和分析的最新进展导致了HCV基因组学的革命。 了解不同的RNA在细胞中发挥的复杂作用。该提案寻求一种分子 了解HCV感染过程中病毒和宿主细胞之间的RNA-蛋白质相互作用， 艺术接近。 在目标1中，我将分析HCV双功能蛋白酶/RNA解旋酶NS3的体内RNA靶点。虽然 NS3是目前DAA治疗的靶点，没有研究分析NS3解旋酶的RNA底物 活动是。据推测，NS3靶向病毒RNA，但不知道解旋酶是否影响细胞内的 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 识别直接与蛋白质相互作用的RNA区域，我将使用各种突变体和/或 药物来探测改变解旋酶或蛋白酶活性的后果。 在目标2中，我将测试HCV RNA基因组是否与细胞蛋白相互作用。派尔实验室发现 正常HCV复制和感染性所需的保守RNA结构。我将使用RAP-MS 揭示宿主细胞蛋白质是否与这些结构相互作用。后续实验将证实 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学。 这项工作的最终目标是帮助DAA的发展，发现HCC进展的标志物， 发现感染的各个方面，可以为其他RNA病毒的研究提供信息。

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.