Identifying cellular long non-coding RNAs as targets for HIV intervention

识别细胞长非编码 RNA 作为 HIV 干预的靶点

• 批准号: 9334104
• 负责人: Xinxia Peng
• 金额: $ 24.14万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-17 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9334104
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Antisense Oligonucleotides; Binding; Biological; Biological Process; Biology; CD4 Positive T Lymphocytes; Cell Nucleus; Cells; Code; Collaborations; Coupling; Custom; Data; Data Set; Disease; Epidemic; Exons; Future; Gene Expression; Genetic Transcription; Genome; Genomics; Goals; HIV; HIV Infections; HIV therapy; HIV-1; Human; Human Genome; Immune; Immune response; Individual; Infection; Integration Host Factors; Intervention; Investigation; Length; Libraries; Life Cycle Stages; Methods; Nucleotides; Pathway interactions; Play; Primates; Proteins; Publishing; RNA; RNA Sequences; Regulator Genes; Role; Series; Small Interfering RNA; System; Techniques; Technology; Testing; Tissues; Transcript; Untranslated RNA; Virus; Virus Diseases; Virus Replication; computational network modeling; deep sequencing; design; differential expression; in vivo; inhibitor/antagonist; insight; knock-down; novel; therapeutic target; transcriptome; transcriptome sequencing

PROJECT SUMMARY Since the beginning of the AIDS epidemic 35 years ago, much has been learned about how HIV replicates in human immune cells and how persistent HIV infection causes AIDS. However, questions still remain, including the molecular interactions regulating HIV replication in human cells and which interactions can be targeted in order to cure HIV/AIDS. Recent advances in RNA technologies have led to the discovery of an extraordinary number of long non-coding RNAs (lncRNAs) in the human genome. While the functions of these lncRNAs are largely unknown, individual studies show that lncRNAs can act as regulators of gene expression, positively or negatively, through diverse mechanisms. Recently, we used transcriptome deep sequencing (RNAseq) analysis of HIV-1 infected human CD4+ T cells to identify human lncRNAs that are differentially expressed during HIV infection. We then computationally predicted the functions of these lncRNAs. Our preliminary studies now show that HIV-1 replication in human CD4+ T cells is perturbed when we knocked down the expression of some of the identified lncRNAs. Here, we propose using a systems approach to better understand the roles of cellular lncRNAs in HIV infection and to discover candidate lncRNAs as targets for HIV intervention. This project includes two Specific Aims: 1) prioritize cellular lncRNAs involved in HIV-1 infection of human CD4+ T cells; and 2) experimentally validate candidate lncRNAs as targets for inhibiting HIV replication. In Aim 1, we propose to integrate two complementary high-throughput methods to identify the most relevant lncRNAs. In collaboration, we will conduct a large lncRNA siRNA library screen to identify lncRNAs required for HIV-1 infection. We will also expand RNAseq analysis of lncRNAs across additional conditions. In particular, we will treat HIV-1 infected CD4+ T cells with a series of well-known inhibitors targeting different stages of HIV life cycle, to uncover lncRNAs that are involved in inhibiting HIV-1 replication or that are essential for specific stages of HIV replication cycle. Using information gained from these two approaches, we will perform a comprehensive lncRNA function prediction and candidate prioritization utilizing additional orthogonal datasets. In Aim 2, we will first evaluate the impact of highly ranked candidate lncRNAs on HIV-1 replication in human CD4+ T cells, by knocking down lncRNA expression with targeted antisense oligonucleotides. For selected lncRNAs that significantly inhibit HIV-1 replication, we will identify key pathways and biological functions involved in the inhibition. These will be done by coupling computational network modeling with RNAseq analysis which will determine expression changes induced by lncRNA perturbation. We will also evaluate these lncRNAs with additional mechanistic studies including lncRNA subcellular localization and mapping lncRNA interacting proteins and functional subsequences within lncRNA. Together, these analyses will allow us to better understand the functions of lncRNAs and their role in HIV replication, and to identify specific lncRNAs as novel targets for HIV intervention.

项目摘要 自从35年前艾滋病开始流行以来，人们对艾滋病毒如何在体内复制已经有了很多了解。 人类免疫细胞以及持续的HIV感染如何导致艾滋病。然而，问题仍然存在，包括 调节人类细胞中HIV复制的分子相互作用，以及哪些相互作用可以靶向于 为了治愈艾滋病。RNA技术的最新进展导致了一个非凡的发现， 人类基因组中长链非编码RNA（lncRNA）的数量。虽然这些lncRNA的功能是 在很大程度上未知，个别研究表明lncRNA可以作为基因表达的调节因子，积极或 消极地，通过各种机制。最近，我们使用转录组深度测序（RNAseq） 分析HIV-1感染的人CD 4 + T细胞以鉴定差异表达的人lncRNA 在艾滋病毒感染期间。然后我们通过计算预测了这些lncRNA的功能。我们的初步 现在的研究表明，当我们敲低HIV-1在人类CD 4 + T细胞中的复制时， 一些已鉴定的lncRNA的表达。在这里，我们建议使用系统方法，以更好地 了解细胞lncRNA在HIV感染中的作用，并发现候选lncRNA作为HIV的靶点 干预该项目包括两个具体目标：1）优先考虑参与HIV-1感染的细胞lncRNA 人CD 4 + T细胞;和2）实验验证候选lncRNA作为抑制HIV的靶标 复制的在目标1中，我们建议整合两种互补的高通量方法，以确定最 相关lncRNA。在合作中，我们将进行一个大型lncRNA siRNA文库筛选，以识别lncRNA HIV-1感染所必需的。我们还将在其他条件下扩展lncRNA的RNAseq分析。在 特别是，我们将用一系列众所周知的抑制剂治疗HIV-1感染的CD 4 + T细胞，这些抑制剂靶向不同的细胞， HIV生命周期的各个阶段，以揭示参与抑制HIV-1复制或 对HIV复制周期的特定阶段至关重要。利用这两种方法获得的信息，我们 将利用额外的技术进行全面的lncRNA功能预测和候选物优先排序。 正交数据集在目标2中，我们将首先评估高排名候选lncRNA对HIV-1的影响， 通过用靶向反义寡核苷酸敲低lncRNA表达，在人CD 4 + T细胞中复制 寡核苷酸对于选定的显著抑制HIV-1复制的lncRNA，我们将确定关键途径， 以及抑制中涉及的生物学功能。这些将通过耦合计算网络来完成 用RNAseq分析建模，其将确定由lncRNA扰动诱导的表达变化。我们 还将通过其他机制研究（包括lncRNA亚细胞定位）评估这些lncRNA 并绘制lncRNA相互作用蛋白和lncRNA内的功能性序列。所有这些 分析将使我们更好地了解lncRNA的功能及其在HIV复制中的作用， 鉴定特异性lncRNA作为HIV干预的新靶点。