Virological and immunological mechanisms of hepatitis C virus persistence

丙型肝炎病毒(丙型肝炎病毒)感染的特点是持久性高。尽管这一现象证明了免疫逃避的病毒策略，但低效病毒控制的分子机制仍然很不清楚。在第一个资助期间，我们建立了一个模拟病毒持久性的细胞培养系统，并获得了丙型肝炎病毒可能利用干扰素(干扰素)反应的随机性的证据。此外，我们还证明了非结构蛋白5A的结构域2以序列依赖和LGP2依赖的方式抑制由RIG-I和MDA5触发的干扰素α的诱导。通过基于小干扰RNA的筛选，我们确定了负责干扰素α和干扰素γ介导的抑制丙型肝炎病毒复制的效应蛋白。最后，通过使用活细胞成像，我们发现丙型肝炎病毒以干扰素α依赖的方式触发应激颗粒的诱导。它们的组装和分解是高度动态的，这对细胞的生存和持久性非常重要。在下一个资助期，我们的目标是继续描述对持久性至关重要的病毒和细胞条件。我们将遵循两个相辅相成的项目。第一个目的是了解干扰素反应的动态及其对病毒传播的影响。在上述结果的基础上，我们将继续我们的研究，使用活细胞成像来分析干扰素应答的随机性及其与病毒传播的关系。一个警告是，丙型肝炎病毒允许的细胞系不产生干扰素，因此，干扰素的反应只能在外源细胞因子添加的情况下进行研究。因此，这个子项目的一个重要目标是建立能够产生内源性干扰素的丙型肝炎病毒允许细胞系，从而更接近体内的情况。在任一系统中产生的数据被用来建立和验证描述干扰素应答的随机性的数学模型，并预测丙型肝炎病毒感染的结果，即持久性或病毒消除。重要的是，结果将通过丙型肝炎病毒感染患者的肝脏活检和单细胞分析来验证。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在丙型肝炎病毒介导的干扰素反应激活和NS5A抑制反应中的作用。在这里，我们将从两个方面进行研究：第一，MDA5对丙型肝炎病毒的识别机制是什么，LGP2对其有何增强作用？第二，NS5A抑制干扰素产生的机制是什么？这些研究的结果将有助于更好地理解丙型肝炎病毒用来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.