Development of real time fluorescence-based assays for elucidating HCV entry dyna

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)可引起急、慢性肝炎和肝细胞癌。值得注意的是，丙型肝炎病毒在肝癌的上升中发挥了重要作用，占美国病例的50%。然而，尽管存在明显的需求，目前还没有疫苗可用于预防丙型肝炎病毒感染，而且只有少数慢性感染患者对当前的治疗方案有反应。因此，显然迫切需要新的有效的丙型肝炎病毒抗病毒药物。特别是，加入丙型肝炎病毒代表着一个有希望的多方面的药物发现机会；然而，仍然需要对这一过程进行更深入的了解和连贯的生化描述，以促进这种努力。重要的是，随着最近第一个强大的细胞培养丙型肝炎病毒感染系统的发展，现在有可能研究丙型肝炎病毒进入的分子细节。因此，R03提案的目标是开发用于研究丙型肝炎病毒进入的活细胞成像研究技术和方法，方法是采用已在其他病毒系统中成功使用的荧光标记策略，用于新的丙型肝炎病毒细胞培养感染系统。因此，该建议的具体目的是：1)建立一种荧光去猝灭的实时检测方法，用于阐明体外丙型肝炎病毒与细胞融合的动力学；2)建立一种基于荧光的单病毒跟踪实时检测方法，用于监测丙型肝炎病毒在体外进入和转运的早期事件。这两种不同但互补的方法将进一步加深我们对丙型肝炎病毒进入动力学的理解，并为开发新的丙型肝炎病毒抑制剂铺平道路。 公共卫生相关性：丙型肝炎病毒(丙型肝炎病毒)感染全球超过1.8亿人，导致急慢性肝炎和肝细胞癌，但目前没有保护性疫苗可用，只有一小部分受感染的患者对当前的治疗方案有反应。为了设计更有效的抗病毒药物，关键是要研究丙型肝炎病毒的生命周期，以了解感染的动力学，并确定哪些感染步骤代表有效的抗病毒靶点。由于进入丙型肝炎病毒代表了一个有希望的多方面的药物发现机会，我们建议开发和使用基于荧光的实时分析来研究调节病毒进入的动力学和条件。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) causes acute and chronic hepatitis and hepatocellular carcinoma (HCC). Notably, HCV has played a major role in the rise of HCC, accounting for 50% of cases in the United States. Despite an obvious need however, no vaccine is available to protect against HCV infection and only a subset of chronically infected patients respond to current treatment options. As such, there is an obvious and immediate need for new effective HCV antivirals. In particular, HCV entry represents a promising multi-faceted opportunity for drug discovery; however a deeper understanding and coherent biochemical description of the process is still needed to facilitate such endeavors. Importantly, with the recent development of the first robust cell culture HCV infection system, the ability to investigate the molecular details of HCV entry is now possible. As such, the objective of this R03 proposal is to develop live cell imaging research technologies and methodologies for the study of HCV entry by adapting fluorescent labeling strategies that have been successfully utilized in other viral systems for use with the new HCV cell culture infection system. Accordingly, the specific aims of this proposal are: 1) To develop a fluorescence dequenching real-time assay for elucidating the dynamics of HCV-cell fusion in vitro and 2) To develop a fluorescence-based single- virus tracking real-time assay for monitoring the early events of HCV entry and trafficking in vitro. These two different but complimentary approaches will further our understanding of HCV entry dynamics and pave the way for the development of new HCV inhibitors. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infects more than 180 million people worldwide, causing acute and chronic hepatitis and hepatocellular carcinoma, however no protective vaccine is available and only a subset of infected patients respond to current treatment options. To design more effective antivirals it is crucial to study the HCV lifecycle to understand the dynamics of infection and identify which steps of infection represent effective antiviral targets. Because HCV entry represents a promising multi-faceted opportunity for drug discovery, we propose to develop and use fluorescence-based real-time assays to investigate the kinetics and conditions that regulate viral entry.