Studies of hepatitis C virus polarized cell entry and host factor requirements

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是美国肝脏疾病的主要原因。由于没有特定的抗丙型肝炎病毒疗法，目前使用的基于干扰素的治疗是不够的，因为它有严重的副作用，而且只对一半主要基因感染的人有效。我过去、现在和将来的研究旨在了解丙型肝炎病毒的复制机制，最终目标是发现新的抗病毒靶点。在过去，我研究了丙型肝炎病毒复制是如何调控的，病毒蛋白如何相互作用以及细胞因素，并帮助建立了在细胞培养中研究丙型肝炎病毒的系统。目前，我的研究集中在病毒生命周期中涉及进入宿主细胞的最早事件，这些过程知之甚少。特别是，尽管有几个宿主因素被认为与丙型肝炎病毒的进入有关，但人们对它们是如何被病毒利用的知之甚少。我们最近发现的紧密连接蛋白claudin-1(CLDN1)在这个过程中是必不可少的，这是该领域的一个重大进展。这一发现开启了对丙型肝炎病毒细胞进入的全新看法，因为CLDN1参与细胞极性的研究强烈表明，肝细胞的极化性质可能会影响丙型肝炎病毒进入细胞的方式。该建议描述了以下实验：1)研究丙型肝炎病毒进入极化细胞，2)确定病毒粒子如何与已知的丙型肝炎病毒进入因子相互作用和利用，以及3)对能够使人和小鼠细胞感染丙型肝炎病毒的丙型肝炎病毒进入因子进行额外的筛选。这样的研究将提供对丙型肝炎病毒复制的更多、更必要的洞察力，并为未来对丙型肝炎病毒和其他相关病毒复制的研究奠定基础，我将在作为学术研究实验室负责人的职业生涯的独立阶段进一步研究这一问题。丙型肝炎病毒所致的肝功能衰竭是肝移植的主要原因，由于肝移植后普遍再感染，导致肝移植失败，常常导致肝纤维化的快速进展和随后的肝移植失败。这些实验旨在进一步了解丙型肝炎病毒的进入过程，与开发新的抗病毒疗法直接相关，以抑制丙型肝炎病毒细胞进入，从而防止移植物再感染，从而极大地提高肝移植的效果。此外，这项工作与发展急需的丙型肝炎病毒小动物模型直接相关。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is the leading cause of liver disease in the United States. With no specific anti-HCV therapies, the currently employed interferon-based treatment is inadequate, as it has severe side effects and is only effective in half of the major genotype infected individuals. My past, current, and future research is aimed at understanding the HCV replication mechanisms, with the ultimate goal of uncovering novel antiviral targets. In the past, I have studied how HCV replication is regulated, how viral proteins interact with each other and cellular factors, and helped establish systems to study HCV in cell culture. Currently, my research is focused on the earliest events in the viral life cycle involving entry into the host cell, which are poorly understood processes. In particular, although several host factors have been implicated as involved in HCV entry, little is know as to how they are utilized by the virus. Our recent identification of the tight junction protein, claudin-1 (CLDN1), as essential for this process was a major development in this field. This discovery has opened-up a whole new view of HCV cell entry, as the involvement of CLDN1 in cell polarity strongly suggests that the polarized nature of an hepatocyte may influence how HCV enter a cell. This proposal describes experiments to: 1) study HCV entry into polarized cells, 2) define how the virion interacts with and utilizes the known HCV entry factors, and 3) perform additional screens for HCV entry factors able to render both human and murine cells infectable with HCV. Such investigations will provide greater, much needed insight in HCV replication, as well as lay the foundation for future studies of replication of HCV and other related viruses, which I will pursue further in the independent phase of my career as the head of an academic research laboratory. Liver failure from HCV is the leading cause of liver transplantation, which is often unsuccessful due to universal reinfection after transplantation, frequently resulting in rapid fibrosis progression and subsequent graft failure. The proposed experiments, aimed at further understanding the HCV entry process, are directly related to the development of novel antiviral therapies to inhibit HCV cell entry, which could prevent graft reinfection and thus greatly improve the effectiveness of liver transplantation. In addition, this work is directly related to the development of much needed HCV small animal models.