Hepatitis C virus polarized cell entry pathways

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是西半球肝癌的主要原因。超过1.7亿人感染了这种病毒，因此肝损伤和癌症的风险增加。丙型肝炎病毒感染是可以治愈的，因此这种健康后果是完全可以避免的。然而，目前的丙型肝炎治疗方法是不够的，因为它与严重的副作用有关，而且只对大约一半的治疗患者有效。有效的丙型肝炎病毒治疗将需要针对多个丙型肝炎病毒复制步骤的抗病毒药物，这些步骤可以组合使用，以避免产生耐药病毒。丙型肝炎病毒的细胞进入过程是抗病毒开发的一个有吸引力的靶点。抑制病毒生命周期的这一阶段将减少丙型肝炎病毒在体内的持久性，这需要不断感染新细胞，以及对其他丙型肝炎病毒抑制剂产生抗药性的病毒突变株的传播。这项申请中提出的研究将促进我们对丙型肝炎病毒细胞进入机制的理解，最终目标是发现新的抗病毒靶点。虽然我们现在知道许多宿主因素是丙型肝炎病毒进入细胞所必需的，但我们不知道它们是如何介导丙型肝炎病毒感染的。本申请中描述的实验是基于这样的假设，即传入的丙型肝炎病毒粒子以顺序的方式利用这些细胞因子中的每一个来调节细胞进入。由于丙型肝炎病毒进入细胞所需的两种宿主蛋白是紧密连接蛋白，调节这些蛋白的定位和功能的细胞极性可能会影响丙型肝炎病毒的细胞进入过程。虽然大多数丙型肝炎病毒进入细胞的研究都是在非极化细胞中进行的，但我们最近开发了第一个极化细胞系统，该系统可以有效地感染在细胞培养中生长的真实丙型肝炎病毒。这 该系统将作为本申请中提出的研究的基础。具体地说，我们建议使用多种丙型肝炎病毒细胞进入因子特异性抑制剂来比较非极化和极化状态下的丙型肝炎病毒细胞进入途径。此外，我们将测试调节细胞极性如何影响丙型肝炎病毒细胞进入，并通过进入因子特异性抑制剂、沉默和与各种突变体的互补来检查宿主因素要求。我们还将通过分析进入因子特异性抑制物的作用动力学，并可视化非极化和极化细胞中的丙型肝炎病毒细胞进入过程，来分析每个进入因子何时何地被使用。最后，我们将确定宿主因子和传入的丙型肝炎病毒粒子之间的物理和遗传相互作用，并探索这些相互作用如何受到细胞极性的影响。

DESCRIPTION (provided by applicant): The hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. Over 170 million people are infected with this virus and thus at increased risk of liver damage and cancer. HCV infection is curable, and thus such health consequences are completely avoidable. However the current HCV therapy is inadequate, as it is associated with severe side effects and only effective in about half of treate individuals. Effective HCV treatment will require antivirals targeting multiple HCV replication steps that can be used in combination to avoid the derivation of resistant viruses. The HCV cell entry process is an attractive target for antiviral development. Inhibiting this stage of the virallife cycle would curtail the persistence of HCV in vivo, which requires constant infection of new cells, as well as the spread of viral mutants that have developed resistance to other HCV inhibitors. The research proposed in this application will advance our understanding of HCV cell entry mechanisms, with the ultimate goal of uncovering novel antiviral targets. Although we now know that numerous host factors are required for HCV cell entry, we do not understand how they mediate HCV infection. The experiments described in this application are based on the hypothesis that the incoming HCV virion utilizes each of these cellular factors in a sequential manner to mediate cell entry. As two of the host proteins that are required for HCV cell entry are tight junction proteins, it is likely that cell polarity, which regulates the localization and funcions of these proteins, impacts the HCV cell entry process. Although the majority of HCV cell entry studies have been conducted in non-polarized cells, we have recently developed the first polarized cell system that permits efficient infection of authentic HCV grown in cell culture. This system will serve as the foundation for the studies proposed in this application. Specifically, we propose experiments to compare HCV cell entry pathways in non-polarized and polarized using a variety of HCV cell entry factor specific inhibitors. Furthermore, we will test how modulating cell polarity influences HCV cell entry, and examine the host factor requirements with entry factor specific inhibitors, silencing, and complementation with a variety of mutants. We will also analyze when and where each entry factor is used by analyzing the kinetics of action of entry factor specific inhibitors and visualizing the HCV cell entry process in non-polarized and polarized cells. Finally, we will identify physical and genetic interactions between host factors and the incoming HCV virion, and explore how such interactions are affected by cell polarity.