The role of innate immune evasion in hepatitis C virus infection

描述（由申请人提供）：丙型肝炎病毒（HCV）是一种RNA病毒，慢性感染约3%的世界人口，导致约20%的感染者发生肝硬化和/或肝细胞癌。目前基于干扰素-1和利巴韦林的治疗未能治愈大多数慢性感染基因型1型HCV的人，并且对新的直接作用的抗病毒药物的快速出现的耐药性可能成为一个重要问题。相比之下，急性感染的治疗预后要好得多，至少20%的急性HCV病例在没有干预的情况下自发消退。因此，更好地了解在急性期哪些先天免疫途径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，HCV已经进化为逃避先天性和适应性免疫系统。在病毒感应的最早阶段，HCV NS 3 -4A蛋白酶切割两个衔接分子MAVS和TRIF，使RIG-I和TLR 3途径无效并阻止干扰素的诱导。为了干扰干扰素应答的下游，HCV激活PKR，其通过激活eIF 21导致干扰素刺激基因（ISG）的翻译下调。这些逃避策略的相对重要性和相互作用尚不清楚。HCV的研究一直受到缺乏强大的体外培养系统的原代肝细胞和限制的病毒嗜性，以人类和黑猩猩。虽然鼠肝细胞不能支持HCV感染，但具有肝损伤的免疫缺陷小鼠可以异种移植随着时间的推移而扩增并允许HCV感染的人肝细胞。我们建议使用两个最近开发的体外培养系统来研究RIG-I和TLR 3病毒传感途径和PKR活化在原代人肝细胞中的相对贡献，并在基于FAH缺陷肝损伤小鼠的新小鼠模型中进一步研究这些途径。为了研究RIG-I和TLR 3途径，将用NS 3 -4A切割抗性形式的MAVS和TRIF转导肝细胞。将使用敲低方法和用eIF 21突变体转导原代肝细胞来研究PKR的作用。感染后，将测量病毒传播、持久性、IRF 3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优先考虑哪些免疫调节疗法应该最好地进入临床开发。

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.