Defining the lncRNA-mediated cis regulatory networks that control HIV life cycle in T cells

定义控制 T 细胞中 HIV 生命周期的 lncRNA 介导的顺式调控网络

• 批准号: 9322477
• 负责人: Saba Valadkhan
• 金额: $ 19.81万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9322477
• 关键词: APOCEC3G gene; Address; Anti-Retroviral Agents; Architecture; Biogenesis; Biological Assay; Biological Models; CCL4 gene; CD4 Positive T Lymphocytes; Cell physiology; Cells; Clinical; Code; Complement; Data; Epigenetic Process; Flow Cytometry; Gene Expression Profiling; Genes; Genetic Transcription; Genomics; Goals; HIV; HIV Infections; High-Throughput Nucleotide Sequencing; Hour; Infection; Laboratories; Lead; Life; Life Cycle Stages; Measures; Mediating; Methods; Modeling; Molecular Conformation; NuRD complex; Play; Population; Positioning Attribute; Primary Infection; Process; Proteins; Proviruses; RNA; Receptor Activation; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Surface; System; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Time; Transcriptional Regulation; Transgenes; Untranslated RNA; Validation; Viral Genome; Viral Load result; Viral load measurement; Virus Diseases; Virus Latency; base; differential expression; epigenetic regulation; knock-down; loss of function; new therapeutic target; novel; overexpression; programs; promoter; protein expression; response; small hairpin RNA; therapeutic target; transcriptome; transcriptome sequencing; transcriptomics; validation studies; viperin

Abstract The broad, long term goal of the proposed research is to identify new therapeutic targets against HIV through the study of long non-coding RNAs (lncRNAs), a novel class of genes that due to their relatively recent discovery, have remained largely unstudied. Despite the availability of highly effective anti-retroviral therapies, eradication of HIV has remained impossible. This is largely due to the inability of existing therapies to target the latent reservoir of transcriptionaly silent HIV proviruses. While our understanding of HIV latency is far from complete, the existing data point to an important role for transcriptional and epigenetic regulatory programs in HIV latency. Interestingly, emerging data indicate that lncRNAs play critical roles in both transcriptional and epigentic regulation, pointing to the potential of this class of cellular RNAs as therapeutic targets against HIV. As a first step toward addressing this exciting possibility, we have taken a high throughput sequencing approach to define the extent of involvement of lncRNAs in HIV life cycle, targeting the clinically crucial steps of early infection, latency and reactivation. Our preliminary studies have revealed that all the above-listed stages of HIV life cycle are associated with differential expression of a large number of lncRNAs. Some of the differentially expressed lncRNAs originated from genomic loci that were positioned in close proximity of protein coding genes with key roles in HIV biogenesis. Intriguingly, in some cases, the architecture of the locus pointed to the possibility of a regulatory interaction between the lncRNA and the protein-coding RNA. Validation studies on two such loci proved the strong regulatory impact of the lncRNAs on their protein-coding neighbors. These exciting results led to the hypothesis that such local, "cis" regulatory networks may play an important functional role in HIV biogenesis and lead to identification of novel therapeutic targets. We propose to address this hypothesis under three specific aims. In Aim 1, we will complement our existing high throughput sequencing approach by including additional stages of HIV life cycle, including earlier time points of primary infection, and early and intermediate stages of latency and proviral reactivation. These studies will not only reveal the true extent of involvement of lncRNAs in HIV life cycle, but also they are likely to provide additional examples of potential cis-regulatory networks operating in HIV biogenesis. In Aim 2, we will test the validity of the potential cis-regulatory loci identified in Aim 1 using RT-PCR-based methods after forcefully increasing or decreasing the level of the lncRNAs in each locus using shRNA or transgene-mediated overexpression methods. Finally, in Aim 3, we will determine whether the validated cis-regulatory relationships impact HIV biogenesis. The most promising candidates with the strongest impacts on HIV biogenesis will be selected for additional studies to further evaluate their potential as anti-HIV therapeutic targets. Taken together, this project will define the extent of involvement of lncRNAs in HIV life cycle, and will identify cis regulatory networks that may impact the replication, latency or reactivation of HIV.

抽象的 拟议的研究的广泛，长期目标是通过 长期非编码RNA（LNCRNA）的研究，这是一种新型基因，由于它们相对较新 发现，在很大程度上没有研究。尽管有高效的抗逆转录病毒疗法可用，但 根除艾滋病毒是不可能的。这在很大程度上是由于现有疗法无法针对 转录静音HIV病毒的潜在水库。虽然我们对艾滋病毒潜伏期的理解远非 完成的现有数据指出了转录和表观遗传调节计划的重要作用 艾滋病毒潜伏期。有趣的是，新兴数据表明lncrnas在转录和 表面性调节，指出这类细胞RNA作为针对HIV的治疗靶标的潜力。 作为解决这一激动人心的可能性的第一步，我们采取了高吞吐量测序方法 为了定义LNCRNA参与HIV生命周期的程度，针对早期至关重要的步骤 感染，潜伏和重新激活。我们的初步研究表明，HIV的所有上述阶段 生命周期与大量LNCRNA的差异表达有关。一些差异 表达的lncRNA起源于基因组基因座，其位于蛋白质编码的近端 具有关键作用在HIV生物发生中的基因。有趣的是，在某些情况下，该基因座的结构指向 LNCRNA与蛋白质编码RNA之间进行调节相互作用的可能性。验证研究 两个这样的基因座证明了LNCRNA对其蛋白质编码邻居的强烈调节作用。这些 令人兴奋的结果导致了这样的假设，即这种本地的“顺式”监管网络可能起着重要的功能 在HIV生物发生中的作用并导致对新型治疗靶标的鉴定。我们建议解决这个问题 假设以三个特定目的。在AIM 1中，我们将补充现有的高通量测序 通过包括艾滋病毒生命周期的额外阶段，包括原发性感染的早期时间点以及 潜伏期和病毒重新激活的早期和中间阶段。这些研究不仅会揭示真实 LNCRNA参与HIV生命周期的程度，但它们也可能提供其他例子 潜在的顺式调节网络在HIV生物发生中运作。在AIM 2中，我们将测试 在AIM 1中使用基于RT-PCR的方法在强力增加或 使用shRNA或转基因介导的过表达方法降低每个基因座的LNCRNA水平。 最后，在AIM 3中，我们将确定经过验证的顺式调节关系是否影响HIV生物发生。 将选择对HIV生物发生影响最大的最有希望的候选人 研究进一步评估其作为抗HIV治疗靶标的潜力。综上所述，这个项目将定义 LNCRNA参与HIV生命周期的程度，并将确定可能 影响艾滋病毒的复制，潜伏期或重新激活。