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

这些研究的长期目标是确定在丙型肝炎期间发生的先天宿主与病毒的相互作用 病毒(丙型肝炎病毒)感染，希望这将导致对这种疾病的新的治疗方法。丙型肝炎是一种 引起急、慢性肝炎和肝细胞病变的非细胞病变的正链RNA病毒 癌症。在美国，约有200-400万人慢性感染丙型肝炎病毒，1.7亿人感染丙型肝炎病毒 全世界的人都是慢性感染者，其中许多人会死于肝功能衰竭和肝细胞癌。 癌症。最近发展的一种稳健的丙型肝炎病毒感染细胞培养模型允许分析 整个丙型肝炎病毒生命周期，并有助于分析决定丙型肝炎病毒结局的宿主-病毒相互作用 感染。利用这个模型，我们最近发现，病毒NS3/4A蛋白水解酶阻断了双链 (Ds)RNA信号和通过切割dsRNA信号中的关键中间产物而诱导的1型干扰素(IFN) 途径，我们发现了一种全新的不依赖NS3/4A的机制，通过这种机制，丙型肝炎病毒可以逃避 受感染细胞中的先天宿主反应。我们还发现，干扰素能迅速调节血管紧张素转换酶的表达。 大量的细胞microRNAs(MiRs)，其中几个在丙型肝炎病毒内具有序列预测的靶标 基因组中，与这些干扰素诱导的miR中的几个相对应的合成miR-模拟物复制 干扰素对丙型肝炎病毒感染的抗病毒作用，这些MIR的中和显著降低了抗病毒作用 干扰素抗丙型肝炎病毒的作用。最后，我们发现干扰素抑制肝脏特异性miR-122的表达 已知这对丙型肝炎病毒复制是必不可少的。这些发现确定了一个先前未被怀疑的效应器 帮助控制丙型肝炎病毒感染的固有宿主反应的手臂。在具体目标1中，我们将使用 用分子和生化方法确定介导NS3/4A非依赖性的病毒蛋白(S) 抑制dsRNA诱导的干扰素表达(亚目标1a)，确定细胞内dsRNA信号分子 以(那些)蛋白(S)(亚目标1b)为靶标，并确定这种新宿主逃避的分子基础 机制(次级目标1c)。这些结果将丰富我们对丙型肝炎病毒持续存在的基础的理解。 感染，并潜在地确定丙型肝炎病毒中一个以前未被认识的靶点(S)用于开发抗病毒药物 毒品。在具体目标2中，我们将使用微阵列技术来定义完整的细胞microRNA 由人类肝细胞中的1型和2型干扰素调节(亚目的2a)；确定是否有任何 这些干扰素调节的MIR抑制丙型肝炎病毒感染(子目标2b)，并介导干扰素-S的抗病毒作用(子目标2c)； 并确定这些miRs的病毒和细胞靶点(亚靶2d)，以及它们在病毒生命周期中的步骤(S)。 当它们抑制丙型肝炎病毒感染时中断(亚目的2e)。总而言之，这些实验将提供对 一种全新的先天防御机制，保护宿主免受丙型肝炎病毒感染，并可能导致 开发新的抗病毒策略，利用这一机制终止慢性丙型肝炎病毒感染。

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.