Hepatic differentiation of stem cells and the cellular determinants of hepatitis

描述（由申请人提供）：我们计划的广泛，长期目标是通过表征丙型肝炎病毒（HCV）感染所必需的细胞辅因子来促进病毒-宿主细胞相互作用的知识。HCV感染全球1.7亿人，是肝细胞癌的主要原因。了解该病毒如何与宿主细胞相互作用（例如，是什么使细胞易受HCV感染）对于诊断、抗病毒药物和预防性疫苗的开发至关重要。我们以前的多能干细胞和定向肝分化的研究表明，在分化过程中的肝脏特化和容许HCV感染之间的相关性。从非允许的细胞过渡到允许的表征揭示了参与调节病毒易感性的候选基因。其中一种细胞因子 与HCV糖蛋白E1结合，在HCV生命周期中起重要作用。这里提出的实验将确定在肝脏发育过程中HCV容许性的分子决定因素，并确定新的细胞辅助因子和HCV治疗的潜在药物靶点。将进行以下实验：（1）干细胞中推定的前病毒因子的抑制和推定的抗病毒因子的过表达。随后将进行肝分化和分化的人肝细胞样细胞（DHH）的HCV感染。(2)一种新的前病毒因子的机制研究，其作用已被我们的初步研究所验证。HCV生命周期的具体步骤将被检查，病毒和宿主蛋白之间的相互作用将被解剖。(3)体内实验旨在评估基因修饰的DHHs重新填充小鼠肝脏并赋予嵌合肝脏HCV抗性的潜力。拟议研究的结果不仅将提供对宿主细胞控制HCV易感性的特性的重要见解，而且还揭示了抗病毒干预的新治疗靶点。

DESCRIPTION (provided by applicant): The broad, long-term goal of our program is to advance knowledge of virus-host cell interactions by characterizing cellular cofactors essential for hepatitis C virus (HCV) infection. HCV infects 170 million people worldwide and is major cause of hepatocellular carcinoma. Understanding how this virus interacts with the host cell (e.g., what makes a cell susceptible to HCV infection) is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. Our previous studies of pluripotent stem cells and directed hepatic differentiation demonstrated a correlation between hepatic specification during the differentiation process and permissiveness to HCV infection. Characterization of the cellular transition from nonpermissive to permissive revealed candidate genes that are involved in regulating viral susceptibility. One of these cellular factors interacts with HCV glycoprotein E1 and plays an essential role in the HCV life cycle. The experiments proposed here will determine the molecular determinants of HCV permissiveness during hepatic development and identify new cellular cofactors and potential drug targets for HCV therapy. The following experiments will be performed: (1) Suppression of putative proviral factors and overexpression of putative antiviral factors in stem cells. These will be followed by hepatic differentiation and HCV infection of the differentiated human hepatocyte-like cells (DHHs). (2) Mechanistic studies of a novel proviral factor whose role has been validated by our preliminary studies. Specific steps of the HCV life cycle will be examined, and the interaction between viral and host protein will be dissected. (3) In vivo experiments designed to assess the potential of genetically modified DHHs to repopulate mouse liver and confer HCV resistance to the chimeric liver. The results of the proposed studies will not only provide significant insights into the properties of the host cells that govern HCV susceptibility but also reveal new therapeutic targets for antiviral intervention.