Hepatitis C virus polarized cell entry pathways

描述（由申请人提供）：丙型肝炎病毒（HCV）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。HCV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HCV治疗是不够的，因为它与严重的副作用有关，并且仅在约一半的治疗个体中有效。有效的HCV治疗需要靶向多个HCV复制步骤的抗病毒药物，这些药物可以联合使用，以避免产生耐药病毒。HCV细胞进入过程是抗病毒开发的有吸引力的靶点。抑制病毒生命周期的这一阶段将减少HCV在体内的持续存在，这需要不断感染新细胞，以及对其他HCV抑制剂产生耐药性的病毒突变体的传播。本申请中提出的研究将促进我们对HCV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HCV进入细胞需要许多宿主因子，但我们不了解它们如何介导HCV感染。本申请中描述的实验是基于这样的假设，即进入的HCV病毒体以顺序方式利用这些细胞因子中的每一种来介导细胞进入。由于HCV细胞进入所需的两种宿主蛋白是紧密连接蛋白，因此可能调节这些蛋白的定位和功能的细胞极性影响HCV细胞进入过程。虽然大多数的HCV细胞进入研究已经在非极化细胞中进行，我们最近开发了第一个极化细胞系统，允许有效感染真正的HCV生长在细胞培养物。这 系统将作为本申请中提出的研究的基础。具体而言，我们提出的实验，以比较在非极化和极化使用各种HCV细胞进入因子特异性抑制剂的HCV细胞进入途径。此外，我们将测试如何调节细胞极性影响HCV细胞进入，并检查与进入因子特异性抑制剂，沉默和互补的各种突变体的宿主因子的要求。我们还将通过分析进入因子特异性抑制剂的作用动力学和可视化非极化和极化细胞中的HCV细胞进入过程来分析何时何地使用每个进入因子。最后，我们将确定宿主因素和传入的HCV病毒体之间的物理和遗传相互作用，并探讨这种相互作用是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.