RNA-Protein Interactions During Hepatitis C Virus Infection

项目摘要 丙型肝炎病毒(丙型肝炎病毒)是一种RNA病毒，目前感染着世界3%的人口。通过 丙型肝炎病毒慢性感染肝细胞，导致宿主细胞发生严重的生理变化。这通常会导致 肝硬变，这是导致肝细胞癌的主要原因，需要 肝脏移植。肝癌是全球与癌症相关的死亡的第二大原因，目前 肝癌的治疗是间接的和无效的。尚未成功开发出预防丙型肝炎病毒的疫苗 感染和直接作用抗病毒(DAA)疗法直到最近才取得成功，这在很大程度上要归功于基本的 研究丙型肝炎病毒感染的分子方面的科学研究。 丙型肝炎病毒是一种纯RNA病毒，最近在RNA研究和分析方面的进展导致了一场革命 了解不同的RNA在细胞中扮演的复杂角色。这项提议寻求一种分子 了解丙型肝炎病毒感染过程中病毒与宿主细胞之间的RNA-蛋白质相互作用 艺术走近了。 在目标1中，我将分析丙型肝炎病毒双功能蛋白酶/RNA解旋酶NS3的体内RNA靶标。虽然 NS3是目前DAA治疗的靶点，目前还没有研究分析NS3解旋酶的RNA底物是什么 活动才是。据推测，NS3以病毒rna为靶标，但解旋酶是否影响细胞尚不清楚。 基因表达。为了解决这些未知问题，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，我将使用各种突变体和/或 探索改变解旋酶或蛋白酶活性的后果的药物。 在目标2中，我将测试丙型肝炎病毒RNA基因组是否与细胞蛋白质相互作用。派尔实验室发现了 保守的RNA结构是正常的丙型肝炎病毒复制和传染性所必需的。我将使用RAP-MS来 揭示宿主细胞蛋白质是否与这些结构相互作用。后续实验将直接证实 互动，并分析这些互动的功能。 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开发DAA，发现肝细胞癌进展的标志，以及 发现可以为研究其他RNA病毒提供信息的感染方面。

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.