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

描述（由申请人提供）：丙型肝炎病毒（HCV）可引起急性和慢性肝炎以及肝细胞癌（HCC）。值得注意的是，HCV在HCC的上升中发挥了重要作用，占美国病例的50%。然而，尽管有明显的需求，但没有疫苗可用于预防HCV感染，只有一部分慢性感染患者对目前的治疗方案有反应。因此，存在对新的有效的HCV抗病毒药物的明显且直接的需求。特别是，HCV进入代表了药物发现的一个有前途的多方面的机会;然而，仍然需要对该过程进行更深入的理解和连贯的生物化学描述，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HCV感染系统的发展，现在有可能研究HCV进入的分子细节。因此，本R 03提案的目的是通过调整已成功用于其他病毒系统的荧光标记策略，开发用于研究HCV进入的活细胞成像研究技术和方法，以用于新的HCV细胞培养感染系统。因此，本提案的具体目标是：1）开发用于阐明体外HCV-细胞融合动力学的荧光去猝灭实时测定和2）开发用于体外监测HCV进入和运输的早期事件的基于荧光的单病毒追踪实时测定。这两种不同但互补的方法将进一步加深我们对HCV进入动力学的理解，并为开发新的HCV抑制剂铺平道路。 公共卫生相关性：丙型肝炎病毒（HCV）感染全球超过1.8亿人，引起急性和慢性肝炎和肝细胞癌，但没有保护性疫苗可用，只有一部分感染患者对目前的治疗方案有反应。为了设计更有效的抗病毒药物，研究HCV的生命周期以了解感染的动力学并确定感染的哪些步骤代表有效的抗病毒靶点至关重要。由于HCV进入代表了药物发现的一个有前途的多方面的机会，我们建议开发和使用基于荧光的实时检测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.