Identification and characterization of cellular factors involved in HCV entry

描述（由申请人提供）：这是2006年首次资助的“HCV进入相关细胞因子的鉴定和表征”项目的竞争性更新。我们的实验室已经研究丙型肝炎病毒（HCV）基因表达和复制的机制超过二十年，但我们对病毒进入的理解受到不充分的研究系统的限制。我们和其他人克服了这一障碍，开发了第一个研究HCV体外进入的平台- HCV假颗粒（HCVpp）和完全感染性的HCV细胞培养系统（HCVcc）。随着这些新工具的出现，我们建议确定和表征HCV摄取所需的因素。这些研究的长期目标是利用这些信息创建急需的HCV小动物模型。使用慢病毒为基础的循环重包装方案，我们成功地鉴定了两个紧密连接蛋白，claudin-1和occludin，作为关键的HCV进入因子。我们在这一发现的基础上，阐明了病毒进入小鼠细胞所需的人为因素，现在已经创建了第一个支持整个HCV生命周期的遗传人源化小鼠模型。这一成就为研究HCV在体内的进入以及利用小鼠遗传学的力量对其进行解剖提供了前所未有的机会。我们还在被动和主动疫苗策略中展示了这种动物模型的概念验证。除了推进体内模型外，我们还在寻求更多与病毒生物学相关的细胞培养系统。这些包括原代成人和胎儿肝细胞培养和三维肝类器官。在这一竞争性更新中，我们采用我们的新型体外和体内系统来进一步了解HCV进入所需的病毒-宿主相互作用。我们提出遗传和生物物理方法来揭示摄取的机制。我们的研究将首次确定感染颗粒的组成和结构，探索病毒与宿主相关的脂质代谢因子之间的相互作用，并绘制病毒内化过程中活跃的分子相互作用网络。

DESCRIPTION (provided by applicant): This is a competing renewal of a grant "Identification and characterization of cellular factors involved in HCV entry" first funded in 2006. Our laboratory has been studying the mechanisms of hepatitis C virus (HCV) gene expression and replication for over twenty years, but our understanding of viral entry was limited by inadequate study systems. We and others overcame this hurdle by developing the first platforms for studying HCV entry in vitro - HCV pseudoparticles (HCVpp) and the fully-infectious HCV cell culture system (HCVcc). With the advent of these new tools, we proposed to identify and characterize factors required for HCV uptake. A long- term goal of these studies was to use this information to create much needed small animal models for HCV. Using lentivirus-based cyclic repackaging schemes, we succeeded in identifying two tight junction proteins, claudin-1 and occludin, as critical HCV entry factors. We built on this finding by elucidating the human factors required for viral entry into mouse cells, and have now gone on to create the first genetically humanized mouse model supporting the entire HCV lifecycle. This achievement opens unprecedented opportunities to study HCV entry in vivo and to dissect it using the power of mouse genetics. We have also shown proof-of-concept for this animal model in passive and active vaccine strategies. In addition to advancing in vivo models, we are pursuing more biologically relevant cell culture systems for the virus. These include primary adult and fetal hepatocyte cultures and three-dimensional liver organoids. In this competing renewal, we employ our novel in vitro and in vivo systems to further understand the virus-host interactions required for HCV entry. We propose genetic and biophysical approaches to uncover the mechanisms of uptake. Our studies will, for the first time, define the composition and structure of the infecting particle, probe the interplay between virus and host- associated lipid metabolism factors, and map the molecular interaction network active during viral internalization.