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和利巴韦林的治疗不能治愈大多数慢性1型HCV感染者，对新的直接作用抗病毒药物的快速耐药性可能成为一个重大问题。相比之下，急性感染治疗的预后要好得多，至少20%的急性HCV病例无需干预即可自行消退。因此，更好地了解哪些先天免疫途径在急性期导致病毒清除可能有助于合理设计未来的免疫调节疗法。为了建立慢性，丙型肝炎病毒已经进化到逃避先天和适应性免疫系统。在病毒感知的最初阶段，HCV NS3-4A蛋白酶会切割两个接头分子，MAVS和TRIF，使RIG-I和TLR3通路失效，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，通过激活eIF21导致干扰素刺激基因（ISGs）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。由于缺乏强大的原代肝细胞体外培养系统，以及病毒对人类和黑猩猩的趋向性受到限制，HCV的研究一直受到阻碍。虽然小鼠肝细胞不能支持HCV感染，但肝损伤的免疫缺陷小鼠可以异种移植人肝细胞，随着时间的推移，人肝细胞会扩大，并允许HCV感染。我们建议使用最近开发的两种体外培养系统来研究RIG-I和TLR3病毒感知途径以及PKR激活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 - 1和TLR3通路，肝细胞将被NS3-4A抗切割形式的MAVS和TRIF转导。PKR的作用将通过敲低方法和eIF21突变体原代肝细胞的转导来研究。感染后检测病毒传播、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。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进入临床发展。

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. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is able to establish chronic infection in about 80% of exposed individuals and is the leading cause of end-stage liver disease and liver cancer in the U.S. Current treatments cure less than half of those infected and we therefore propose to investigate the mechanisms by which HCV evades the immune system in primary liver cell cultures and in mice infected with HCV. These studies may guide us in the search of drugs that can activate the immune system to clear HCV infection.