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

描述（由申请人提供）：丙型肝炎病毒（HCV）是美国肝脏疾病的主要原因。由于没有特异性的抗hcv疗法，目前采用的基于干扰素的治疗是不充分的，因为它有严重的副作用，并且只对一半的主要基因型感染者有效。我过去、现在和未来的研究旨在了解HCV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HCV的复制是如何被调节的，病毒蛋白如何相互作用以及细胞因子，并帮助建立了HCV在细胞培养中的研究系统。目前，我的研究主要集中在病毒生命周期中涉及进入宿主细胞的最早事件，这是一个知之甚少的过程。特别是，虽然有几个宿主因素被认为参与了HCV的进入，但对于病毒如何利用它们却知之甚少。我们最近鉴定的紧密连接蛋白CLDN1在这一过程中是必不可少的，这是该领域的一个重大进展。这一发现开辟了HCV进入细胞的全新视角，因为CLDN1参与细胞极性强烈提示肝细胞的极化性质可能影响HCV进入细胞的方式。本提案描述了以下实验：1)研究HCV进入极化细胞，2)定义病毒粒子如何与已知的HCV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HCV的HCV进入因子进行额外筛选。这样的调查将为HCV复制提供更大的、急需的见解，并为未来HCV和其他相关病毒复制的研究奠定基础，我将在独立的学术研究实验室负责人的职业生涯中进一步追求。HCV引起的肝功能衰竭是肝移植的主要原因，由于移植后普遍的再感染，肝移植往往不成功，经常导致快速纤维化进展和随后的移植物衰竭。本实验旨在进一步了解HCV的进入过程，与开发抑制HCV细胞进入的新型抗病毒疗法直接相关，从而防止移植物再感染，从而大大提高肝移植的有效性。此外，这项工作直接关系到急需的HCV小动物模型的开发。

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.