Virological and immunological mechanisms of hepatitis C virus persistence

丙型肝炎病毒（HCV）感染的特点是持久性高。尽管这一现象证明了病毒的免疫逃避策略，但低效病毒控制的分子机制仍然相当不明确。在第一个资助期，我们建立了一个模拟病毒持久性的细胞培养系统，并获得了HCV可能利用干扰素（IFN）反应的随机性的证据。此外，我们证明了非结构蛋白5A的结构域2以序列依赖和lgp2依赖的方式抑制RIG-I和MDA5触发的IFNα的诱导。通过基于siRNA的筛选，我们鉴定了IFNα和ifn γ介导的抑制HCV复制的效应蛋白。最后，通过活细胞成像，我们发现HCV以依赖ifn α的方式触发应激颗粒的诱导。它们的组装和拆卸是高度动态的，这对细胞的生存和持久性很重要。在下一个资助期内，我们的目标是继续我们对持久性所必需的病毒和细胞条件的表征。我们将实施两个互补项目。第一个目的是了解干扰素反应的动力学及其对病毒传播的影响。在上述结果的基础上，我们将继续使用活细胞成像来分析干扰素反应的随机性及其与病毒传播的关系。需要注意的是，允许HCV的细胞系不产生IFN，因此，IFN反应只能在外源性添加细胞因子的情况下研究。因此，本子项目的一个重要目标是建立能够内源性IFN产生的hcv -许可细胞系，从而更接近地模仿体内情况。在这两种系统中产生的数据用于建立和验证描述ifn反应的随机性的数学模型，并预测HCV感染的结果，即持续或病毒消除。重要的是，结果将通过hcv感染患者的肝活检和单细胞分析得到验证。最后，我们的目标是测量活细胞中的T细胞反应，并研究这种反应是否也是随机的。第二个项目涉及MDA5在hcv介导的IFN反应激活和NS5A对其抑制中的作用。我们将从两个方面进行研究：第一，MDA5对HCV的识别机制是什么，LGP2是如何增强HCV的？其次，NS5A抑制IFN产生的机制是什么？这些研究的结果将有助于更好地理解HCV用于克服先天免疫反应的策略。

Hepatitis C virus (HCV) infections are characterized by a high rate of persistence. Although this phenomenon argues for viral strategies of immune evasion, the molecular mechanisms underlying inefficient virus control are still rather poorly defined. During the first funding period we established a cell culture system mimicking viral persistence and obtained evidence that HCV might exploit the stochastic of the interferon (IFN) response. Moreover, we demonstrated that domain 2 of nonstructural protein 5A suppresses induction of IFNα that is triggered by RIG-I and MDA5 in a serial- and LGP2-dependent manner. By using a siRNA based screen we identified effector proteins responsible for IFNα- and IFNγ-mediated suppression of HCV replication. Finally, by using live cell imaging we found that HCV triggers induction of stress granules in an IFNα-dependent manner. Their assembly and disassembly is highly dynamic which is important for cell survival and thus, persistence. In the next funding period we aim to continue our characterization of viral and cellular conditions essential for persistence. We will follow two complementary projects. The first one aims at understanding the dynamics of the IFN response and its impact on virus spread. Building on the results described above we will continue our studies by using live cell imaging to analyze the stochastic of the IFN response and its relation to virus spread. A caveat is that cell lines that are HCV permissive do not produce IFN and therefore, IFN response can only be studied upon exogenous addition of the cytokine. Thus, an important aim of this subproject is to establish HCV-permissive cell lines that are capable of endogenous IFN production, thus mimicking more closely the in vivo situation. Data generated in either system are used to set up and validate a mathematical model describing the stochastic of the IFN-response and predicting the outcome of HCV infection, i.e. persistence or virus elimination. Importantly, results will be validated with liver biopsies of HCV-infected patients and single cell analyses. Finally, we aim to measure T cell response in live cells and to study whether this response is also stochastic. The second project deals with the role of MDA5 in HCV-mediated activation of the IFN response and its suppression by NS5A. Here we will study two aspects: first, what is the recognition mechanism of HCV by MDA5 and how is it enhanced by LGP2? Second, what is the mechanism by which NS5A suppresses IFN production? The results of these studies will contribute to a better understanding of the strategies used by HCV to overcome innate immune responses.