Roles for microRNA-122 in hepatitis C virus RNA amplification

描述（由申请人提供）：据估计，全世界有1.7亿人，美国有400万人感染丙型肝炎病毒（HCV）。大多数患者不能解决感染并发展慢性感染，通常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，对50%的感染者无效。因此，迫切需要鉴定可用作抗病毒治疗中的新靶点的病毒或细胞分子。研究发现，HCV结合两个肝脏特异性microRNA分子miR-122，导致病毒基因组前所未有的新上调。在HCV感染的培养细胞或感染的黑猩猩的肝脏中隔离miR-122导致感染性病毒的急剧损失，而不出现耐药病毒。因此，HCV对miR-122的依赖性是病毒的致命弱点，可用于抗病毒干预。本申请提出使用新型反义分子锁核酸（LNA）来研究miR-122在病毒生命周期和胆固醇生物合成中的作用，所述锁核酸可以容易地递送至动物的肝脏，其中其将miR-122隔离在无活性的小双链RNA中。特别是，第一个目标将使用遗传和生物化学方法表征miR-122/HCV复合物中的RNA-RNA相互作用。目的2将检验以下假设：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（如视黄酸诱导基因I）的激活。这些研究将在特异性和全基因组siRNA介导的基因敲除实验中进行。目的3提出检测含有额外3'末端腺苷或鸟苷残基的miR-122的已知亚型对HCV RNA丰度的作用。深度测序分析和疑似核苷酸转移酶的基因敲除将有助于该分析。最终目标将详细描述miR-122调节Insig 1表达的机制，Insig 1是肝脏中胆固醇和脂肪酸代谢的主要负调节因子。具体而言，将检查miR-122介导的特异性Insig 1同种型mRNA中的不同聚腺苷酸化/切割位点的下调。总体而言，该应用将解决有关miR-122在HCV生命周期和胆固醇代谢中功能的基本方面。这些研究的结果将详细说明真核细胞中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.