Mechanisms of immune evasion by hepatitis C virus and their role in establishment of viral persistence

全世界估计有1.7亿人慢性感染丙型肝炎病毒（HCV），在许多情况下导致严重的肝损伤。高持久性的原因尚不完全清楚，但很明显，先天和适应性免疫反应消灭病毒的失败是特别重要的。在第一个资助期，TP3分析了HCV与功能性抗原呈递的干扰以及HCV逃避干扰素-γ反应的情况，干扰素-γ反应是介导非胞质t细胞对HCV反应的中心细胞因子。建立了真实HLA-A2限制性CD8+ t细胞反应的免疫学模型，为研究抗原呈递调节提供了强有力的系统。TP3的数据进一步表明，HCV在IFN-γ存在下可以持续存在，并确定了一些新的候选基因，这些基因可能参与IFN-γ对HCV的反应。在这项工作的继续，我们将分析50个候选基因在细胞群中差异调节的功能作用，在有效的IFN-γ反应下维持高水平的HCV复制。经过验证的候选药物将对其作用机制进行深入研究。这项工作将揭示IFN-γ耐药性对HCV持久性的贡献，并揭示针对HCV的IFN-γ反应和抗病毒效应蛋白的一般调控。第二个项目将解决能够激活先天免疫反应的病毒复制中间体的周转问题。RNA复制产生大量的病毒病原体相关分子模式（PAMPs），如双链RNA，它们被细胞质（rig - 1, MDA5）或内体（TLR3）模式识别受体识别。在持续感染期间，病毒PAMPs必须经历有规律的更新，这尤其说明了病毒诱导的膜囊泡中包含的双链复制中间体。然而，病毒PAMPs的衰变途径尚未确定。因此，我们的工作旨在揭示病毒复制中间体的处置机制，并阐明其在维持病毒持久性中的作用。我们将首先关注含有病毒RNA的外泌体的作用。外泌体最近被发现由含有亚基因组复制子的细胞释放，能够激活浆细胞样树突状细胞（pDC）。病毒诱导的外泌体形成的调节将揭示它们在病毒RNA的常规周转和规避细胞内先天免疫反应激活方面的功能。分析引起急性感染的其他嗜肝病毒的外泌体分泌和pDC激活情况，将进一步阐明外泌体是正链RNA病毒的普遍现象还是与持续感染有关。

An estimated 170 million people worldwide are chronically infected with hepatitis C virus (HCV), leading in many cases to severe liver damage. The reasons for the high rate of persistence are not fully understood yet, but it is clear that the failure of innate and adaptive immune responses to eliminate the virus is of particular importance. During the first funding period TP3 analyzed interference of HCV with functional antigen presentation and escape of HCV from interferon-γ response, the central cytokine mediating noncytolitic T-cell response to HCV. An immunological model for authentic HLA-A2 restricted CD8+ T-cell response was established, providing a powerful system to study modulation of antigen presentation. The data of TP3 furthermore demonstrated that HCV persistence in presence of IFN-γ can be achieved and identified a number of novel candidate genes potentially involved in IFN-γ response to HCV. In continuation of this work we will analyze the functional role of fifty candidate genes differentially regulated in cell populations maintaining high levels of HCV replication in presence of efficient IFN-γ response. Validated candidates will be subjected to in depth studies on their mechanism of action. This work will reveal the contribution of IFN-γ resistance to HCV persistence and shed light on the general regulation of IFN-γ response and antiviral effector proteins targeting HCV. A second project will address the turnover of viral replication intermediates capable of activating innate immune responses. RNA replication generates huge amounts of viral pathogen associated molecular patterns (PAMPs), like double stranded RNA, which are recognized by cytosolic (RIG-I, MDA5) or endosomal (TLR3) pattern recognition receptors. During persistent infections viral PAMPs must undergo a regular turnover, this accounts particularly for double stranded replication intermediates contained in virus induced membrane vesicles. However, the pathway of decay of viral PAMPs has not been identified yet. Our work therefore aims to uncover the mechanism of disposal of viral replication intermediates and clarify its role in maintaining viral persistence. We will initially focus on the role of exosomes containing viral RNA. Exosomes have recently been found to be released by cells harboring subgenomic replicons and are capable of activating plasmacytoid dendritic cells (pDC). Modulation of virus induced exosome formation will reveal their function regarding regular turnover of viral RNA and circumventing activation of intracellular innate immune responses. Analysis of exosome secretion and pDC activation by other liver-tropic viruses causing acute infections will furthermore elucidate whether exosomes are a general phenomenon of positive strand RNA viruses or linked to persistent infections.