Roles for microRNA-122 in hepatitis C virus RNA amplification

描述（由申请人提供）：全球估计有1.7亿人感染丙型肝炎病毒（HCV），美国有400万人感染。大多数患者的感染没有得到解决，并发展为慢性感染，往往导致终末期肝病和肝细胞癌。目前的治疗仅限于利巴韦林和干扰素1的联合治疗。这种疗法昂贵且对50%的感染者无效。因此，迫切需要识别病毒或细胞分子，可作为抗病毒治疗的新靶点。研究发现，HCV结合肝脏特异性microRNA miR-122的两个分子，导致病毒基因组出现前所未有的上调。在hcv感染的培养细胞或受感染黑猩猩的肝脏中隔离miR-122可导致感染性病毒的显著损失，而不会出现耐药病毒。因此，HCV对miR-122的依赖性是病毒的致命弱点，可以探索抗病毒干预。本应用程序提出研究miR-122在病毒生命周期和胆固醇生物合成中的作用，使用一类新的反义分子，锁定核酸（LNAs），可以很容易地递送到动物的肝脏，在那里它将miR-122隔离在非活性的小双工RNA中。特别是，第一个目标将利用遗传和生化方法表征miR-122/HCV复合体中的RNA-RNA相互作用。目的2将验证以下假设：miR-122保护HCV RNA的5'端序列免受核糖核酸酶或RNA修饰酶的降解，或有助于避免双链RNA传感器（如维甲酸诱导基因i）的激活。这些研究将在特异性和全基因组sirna介导的基因敲低实验中进行。目的3旨在研究已知的含有额外3'端腺苷或鸟苷残基的miR-122同工型对HCV RNA丰度的作用。深度测序分析和基因敲除可疑的核苷酸转移酶将有助于这一分析。最终目的将详细描述miR-122调节Insig1表达的机制，Insig1是肝脏中胆固醇和脂肪酸代谢的主要负调节因子。特别是，将研究mir -122介导的特定Insig1亚型mrna中不同聚腺苷化/切割位点的下调。总的来说，这项应用将解决miR-122在HCV生命周期和胆固醇代谢中的基本功能。这些研究的结果将详细说明真核细胞中由microrna介导的基因表达的新机制，并将为抗病毒治疗指明新的途径。

DESCRIPTION (provided by applicant): An estimated 170 million people worldwide and 4 million people in the United States are infected with hepatitis C virus (HCV). The majorities of patients do not resolve the infection and develop chronic infections that often lead to end-stage liver disease and hepatocellular carcinoma. Current treatment is limited to a combination therapy of ribavirin and interferon 1. This therapy is expensive and ineffective in 50% of infected individuals. Thus, there is an urgent need to identify viral or cellular molecules that can be used as novel targets in antiviral therapy. It was discovered that HCV binds two molecules of a liver-specific microRNA, miR-122, resulting in a novel, unprecedented upregulation of the viral genome. Sequestration of miR-122 in HCV-infected cultured cells or in livers of infected chimpanzees leads to a dramatic loss of infectious virus without emergence of resistant virus. Therefore, the dependence of HCV on miR-122 presents an Achilles heel of the virus that can be explored for antiviral intervention. This application proposes to study the roles for miR-122 in the viral life cycle and in cholesterol biosynthesis using a novel class of antisense molecules, locked nucleic acids (LNAs) that can easily be delivered to the liver in animals where it sequesters miR-122 in an inactive small duplex RNA. In particular, the first aim will characterize the RNA-RNA interactions in the miR-122/HCV complex, using genetic and biochemical approaches. Aim 2 will test the hypothesis that miR-122 protects the 5' end sequences of the HCV RNA from degradation by ribonucleases or RNA modification enzymes, or aids in the avoidance of activation of double-stranded RNA sensors such as the retinoic acid inducible gene I. These studies will be performed in specific and genome-wide siRNA-mediated gene knockdown experiments. Aim 3 proposes to examine roles for the known isoforms of miR-122 that contain extra 3' terminal adenosine or guanosine residues, on HCV RNA abundance. Deep sequencing analysis and gene knockdown of suspected nucleotidyl transferases will aid in this analysis. The final aim will characterize in detail the mechanism by which miR-122 regulates the expression of Insig1, the major negative regulator of cholesterol and fatty acid metabolism in the liver. In particular, the miR-122-mediated down-regulation of a distinct polyadenylation/cleavage site in a specific Insig1 isoform mRNAs will be examined. Overall, this application will address fundamental aspects about the functions of miR-122 in the HCV life cycle and cholesterol metabolism. The outcomes from these studies will detail novel mechanisms of gene expression mediated by microRNAs in eukaryotic cells and will point to new venues for antiviral therapies.