The role of innate immune evasion in hepatitis C virus infection

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是一种RNA病毒，慢性感染约3%的世界人口，导致约20%的感染者出现肝硬化和/或肝细胞癌。目前基于干扰素-1和利巴韦林的治疗方法无法治愈大多数慢性感染1型丙型肝炎病毒的人，对新的直接作用抗病毒药物迅速出现的耐药性可能会成为一个重大问题。相比之下，急性感染的治疗预后要好得多，至少20%的急性丙型肝炎病例在没有干预的情况下自动痊愈。因此，更好地了解哪些先天免疫途径可以在急性期导致病毒清除，可能有助于未来免疫调节疗法的合理设计。为了建立慢性化，丙型肝炎病毒已经进化成躲避先天免疫系统和获得性免疫系统。在病毒感应的最早步骤中，丙型肝炎病毒NS3-4A蛋白酶裂解两个接头分子MAVS和TRIF，使RIG-I和TLR3途径无效，并阻止干扰素的诱导。为了干扰干扰素反应的下游，丙型肝炎病毒激活PKR，从而通过激活eIF21导致干扰素刺激基因(ISGs)的翻译下调。这些规避策略的相对重要性和相互作用尚不清楚。由于缺乏强大的原代肝细胞体外培养系统，以及对人类和黑猩猩的病毒嗜性受到限制，丙型肝炎病毒的研究一直受到阻碍。虽然小鼠肝细胞不能支持丙型肝炎病毒感染，但免疫缺陷的肝损伤小鼠可以与人肝细胞异种移植，人肝细胞随着时间的推移而扩张，并允许感染丙型肝炎病毒。我们建议使用最近发展的两种体外培养系统来研究RIG-I和TLR3病毒传感通路和PKR激活在原代人肝细胞中的相对贡献，并在基于FAH缺乏性肝损伤小鼠的新的小鼠模型中进一步研究这些通路。为了研究RIG-I和TLR3途径，将NS3-4A裂解抗性形式的MAV和TRIF转导肝细胞。PKR的作用将通过敲除方法和转导eIF21突变体的原代肝细胞来研究。感染后，将测量病毒传播、持久性、IRF3易位和ISG诱导。这些研究有望阐明这些先天免疫途径对原代肝细胞抗病毒免疫的相对贡献。通过这些实验获得的结果可以帮助确定哪些免疫调节治疗应该最好地应用于临床开发。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a RNA virus that chronically infects ~3% of the world population leading to cirrhosis and/or hepatocellular carcinoma in ~20% of those infected. Current treatments based on interferon-1 and ribavirin fail to cure the majority of people chronically infected with genotype 1 HCV, and rapidly emerging resistance to new direct-acting antivirals is likely to become a significant problem. By contrast treatment of acute infection has a far better prognosis, and at least 20% of acute HCV cases spontaneously resolve without intervention. A better understanding of which innate immune pathways lead to viral clearance during the acute phase may therefore aid the rational design of future immunomodulatory therapies. In order to establish chronicity, HCV has evolved to evade both the innate and adaptive immune systems. At the earliest steps of viral sensing, the HCV NS3-4A protease cleaves two adaptor molecules, MAVS and TRIF, rendering both RIG-I and TLR3 pathways ineffective and preventing the induction of interferons. In order to interfere downstream from the interferon response, HCV activates PKR, which leads to translational down-regulation of interferon stimulated genes (ISGs) through activation of eIF21. The relative importance and interplay of these evasion strategies is not understood. Research of HCV has been hampered by a paucity of robust in vitro culture systems of primary hepatocytes and by restriction of viral tropism to humans and chimpanzees. While murine hepatocytes cannot support HCV infection, immunodeficient mice with liver injury can be xenografted with human hepatocytes that expand over time and allow for infection with HCV. We propose to use two recently developed in vitro culture systems to investigate the relative contributions of the RIG-I and TLR3 viral sensing pathways and of PKR activation in primary human hepatocytes, and further study these pathways in a new mouse model based on FAH- deficient liver injury mice. To investigate the RIG-I and TLR3 pathways, hepatocytes will be transduced with NS3-4A cleavage-resistant forms of MAVS and TRIF. The role of PKR will be studied using knock down approaches and transduction of primary hepatocytes with eIF21 mutants. After infection, viral spread, persistence, IRF3 translocation and ISG induction will be measured. These studies will hopefully shed light on the relative contributions of these innate immune pathways to antiviral immunity in primary hepatocytes. Results obtained by these experiments can help prioritize which immunomodulatory therapies should best be pursued into clinical development.