Innate Control of Hepatitis C Virus Infection: Antiviral and Evasion Mechanisms

这些研究的长期目标是确定丙型肝炎期间发生的先天宿主-病毒相互作用 病毒（HCV）感染，希望这将导致新的治疗方法，这种疾病。HCV是一种 一种非致细胞病变的正链RNA病毒，可引起急性和慢性肝炎及肝细胞性肝炎 carcinoma.在美国约有2-4百万人慢性感染HCV， 世界范围内有100万人被慢性感染，其中许多人将死于肝功能衰竭和肝细胞癌。 carcinoma.最近开发的HCV感染的稳健细胞培养模型允许分析HCV感染的细胞周期。 整个HCV生命周期，并有助于分析决定HCV结局的宿主-病毒相互作用 感染使用该模型，我们最近表明，病毒NS 3/4A蛋白酶阻断双链 (ds)RNA信号传导和通过切割dsRNA信号传导中的关键中间体的1型干扰素（IFN）诱导 我们发现了一种全新的非NS 3/4A依赖性机制，HCV通过这种机制逃避了免疫抑制。 受感染细胞的先天宿主反应。我们还发现，干扰素可以快速调节 许多细胞微小RNA（miRs），其中一些在HCV内具有序列预测的靶点， 基因组中，合成的miR模拟物对应于这些IFN诱导的miR中的几个， IFN对HCV感染的抗病毒作用，中和这些miR显著降低IFN的抗病毒作用。 IFN对HCV的作用最后，我们发现IFN抑制了肝脏特异性miR-122的表达， 已知其对于HCV复制是必需的。这些发现确定了一个以前未被怀疑的效应器 先天宿主反应的一个分支，有助于控制HCV感染。在具体目标1中，我们将使用 分子和生物化学方法来定义介导非NS 3/4A依赖性的病毒蛋白 抑制dsRNA诱导的IFN表达（子目的1a），鉴定细胞dsRNA信号分子， 被那些蛋白质靶向（subaim 1b），并确定这种新的宿主逃避的分子基础 机制（子目标1c）。这些结果将丰富我们对HCV病毒持续存在的基础的理解 感染，并有可能确定一个以前不受重视的目标（S）在丙型肝炎病毒的发展， 毒品在具体目标2中，我们将使用微阵列技术来定义细胞microRNA的完整组成部分， 在人肝细胞中受1型和2型干扰素调节的蛋白质（子目标2a）;确定是否有任何 这些IFN调节的miR抑制HCV感染（subaim 2b）并介导IFN的抗病毒作用（subaim 2c）; 并鉴定这些miR的病毒和细胞靶点（子目标2d），以及它们在病毒生命周期中的步骤。 当它们抑制HCV感染时中断（子目标2 e）。总的来说，这些实验将提供深入了解 一种全新的先天防御机制，保护宿主免受HCV感染，并可能导致 开发新的抗病毒策略，利用这种机制来终止慢性HCV感染。

The long term goal of these studies is to define the innate host-virus interactions that occur during hepatitis C virus (HCV) infection with the hope that this will lead to novel therapeutic approaches to this disease. HCV is a noncytopathic, positive-strand RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Approximately 2-4 million persons are chronically infected by HCV in the USA and 170 million people are chronically infected worldwide, many of whom will die from liver failure and hepatocelluar carcinoma. The recent development of a robust cell culture model of HCV infection permits analysis of the entire HCV life cycle and facilitates analysis of the host-virus interactions that determine the outcome of HCV infection. Using that model we have recently shown that that the viral NS3/4A protease blocks double stranded (ds) RNA signaling and type 1 interferon (IFN) induction by cleaving a key intermediate in the dsRNA signaling pathway, and we discovered an entirely novel NS3/4A-independent mechanism whereby HCV evades the innate host response in infected cells. We also discovered that IFN rapidly modulates the expression of numerous cellular microRNAs (miRs) several of which have sequence-predicted targets within the HCV genome, that synthetic miR-mimics corresponding to several of these IFN-induced miRs reproduces the antiviral effects of IFN on HCV infection, and that neutralization of these miRs significantly reduces the antiviral effects of IFN against HCV. Finally, we showed that IFN inhibits the expression of the liver-specific miR-122 which is known to be essential for HCV replication. These findings identify a previously unsuspected effector arm of the innate host response that contributes to the control of HCV infection. In Specific Aim 1, we will use molecular and biochemical approaches to define the viral protein(s) that mediate NS3/4A-independent suppression of dsRNA-induced IFN expression (subaim 1a), identify the cellular dsRNA signaling molecule that is targeted by that (those) protein(s) (subaim 1b), and determine the molecular basis of this novel host evasion mechanism (subaim 1c). These results will enrich our understanding of the basis for viral persistence in HCV infection and, potentially, identify a previously unappreciated target(s) in HCV for the development of antiviral drugs. In Specific Aim 2, we will use microarray technology to define the full complement of cellular microRNAs that are regulated by type 1 and type 2 interferons in human liver cells (subaim 2a); determine whether any of these IFN-regulated miRs inhibit HCV infection (subaim 2b) and mediate IFN¿s antiviral effects (subaim 2c); and identify the viral and cellular targets of these miRs (subaim 2d), and the step(s) in the virus life cycle they interrupt when they inhibit HCV infection (subaim 2e). Collectively, these experiments will provide insight into an entirely new innate defense mechanism that protects the host against HCV infection and, potentially, lead to the development of novel antiviral strategies that exploit this mechanism to terminate chronic HCV infection.