Examining the role of cellular miRNAs in HIV-1 Replication

摘要 在美国，估计有120万人感染艾滋病毒。尽管取得了很大进展，但反 逆转录病毒疗法仍然是唯一的治疗选择。ART的长期成功取决于 由于现有药物毒性和ART的出现， 抵抗病毒。细胞miRNAs的抗HIV活性是一个新兴领域，具有很大的潜力， 确定新的治疗靶点。然而，人们对它们之间的分子相互作用知之甚少。 miRNAs和HIV-1。miRNAs是一种小的非编码RNA，通过调节基因表达来负调控基因的表达。 与靶mRNA的3'非翻译区（UTR）结合，导致翻译抑制或mRNA 降解miRNAs在包括抗病毒反应在内的细胞过程的各个方面发挥着重要作用。 已发表的数据表明，细胞miRNA调节许多病毒的复制，包括灵长类泡沫病毒， 病毒、丙型肝炎病毒、爱泼斯坦巴尔病毒、流感病毒和HIV。此应用程序的总体目标是 剖析细胞miRNA赋予抗HIV活性的机制。 细胞miRNAs的抗HIV-1活性首先是通过结合靶点预测发现的， 实验验证从那时起，已经报道了一个抗HIV miRNA家族抑制HIV-1。两 提出了细胞miRNA的抗HIV活性的机制。它们可以与HIV-1转录本结合， 抑制病毒蛋白翻译。或者，它们可以与细胞转录物结合并抑制宿主转录物的翻译。 与病毒复制有关的因素。然而，在理解以下方面存在着关键的知识差距： miRNA介导的抗HIV活性。我们对细胞抗HIV-1 miRNAs的研究旨在填补 这个差距。核心假设是抗HIV-1的miRNAs通过抑制病毒蛋白来抑制HIV-1 抗HIV miRNAs的翻译和调节依赖于DNMT辅助的启动子超表达， 甲基化本提案旨在通过三个具体目标来检验中心假设。目标1： 确定HIV-1翻译的抑制是由抗HIV miRNA与病毒的直接结合介导的， 成绩单目的2：检测DNMT诱导的启动子区甲基化对抗HIV miRNA的调控作用 表情目的3：阐明CpG过度甲基化引起下调的机制。 抗HIV-1 miRNAs。本申请中使用的方法利用了病毒学界面上的新方法， 遗传学、生物化学和分子生物学。我们坚信，这一提案的数据将大大 提高我们对细胞miRNAs抗HIV-1活性的理解，促进新型抗HIV-1药物的开发。 病毒疗法

ABSTRACT In the US, an estimated 1.2 million people are living with HIV. In spite of considerable progress, anti- retroviral therapy (ART) remains the only treatment option. The long-term success of ART is dependent on identification of new drugs against novel therapeutic due to toxicity of current drugs and emergence of ART- resistant viruses. The anti-HIV activity of cellular miRNAs is an emerging area and has a great potential to identify novel therapeutic targets. However, very little is known about the molecular interactions between cellular miRNAs and HIV-1. MiRNAs are small noncoding RNAs that negatively regulate gene expression by binding to the 3’ untranslated region (UTR) of target mRNAs causing translational repression or mRNA degradation. miRNAs play fundamental roles in every aspect of cellular process including anti-viral responses. Published data demonstrate that cellular miRNAs regulate replication of many viruses including Primate foamy virus, Hepatitis C virus, Epstein Barr virus, Influenza virus and HIV. The overall goal of this application is to dissect the mechanism by which cellular miRNAs confer anti-HIV activity. The anti-HIV-1 activity of cellular miRNAs was first discovered by target prediction coupled with experimental validation. Since then a family of anti-HIV miRNAs has been reported to inhibit HIV-1. Two mechanisms are proposed for the anti-HIV activities of cellular miRNAs. They can bind to HIV-1 transcripts and inhibit viral protein translation. Alternatively, they can bind to cellular transcripts and inhibit translation of host factors associated with viral replication. However, there are key knowledge gaps in the understanding of miRNA-mediated anti-HIV activity. Our proposed studies on the cellular anti-HIV-1 miRNAs are designed to fill this gap. The central hypothesis is anti-HIV-1 miRNAs inhibit HIV-1 by repressing viral protein translation and regulation of anti-HIV miRNAs is dependent on DNMT-assisted promoter hyper- methylation. This proposal is organized to test the central hypothesis through three specific aims. Aim 1: To determine that repression of HIV-1 translation is mediated by direct binding of anti-HIV miRNAs to viral transcripts. Aim 2: To test that DNMT-induced promoter hyper-methylation regulates anti-HIV miRNA expression. Aim 3: To elucidate the mechanism by which CpG hyper-methylation causes downregulation of anti-HIV-1 miRNAs. The approach used in this application utilizes novel methods at the interface of virology, genetics, biochemistry, and molecular biology. We strongly believe that data from this proposal will significantly improve our understanding of anti-HIV-1 activity of cellular miRNAs and facilitate development of novel anti- viral therapy.