The spectrum of long non-coding RNAs that regulate HIV expression and latency

调节 HIV 表达和潜伏期的长非编码 RNA 谱系

• 批准号: 10684324
• 负责人: Nadejda S Beliakova-Bethell
• 金额: $ 17.94万
• 依托单位: VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10684324
• 关键词: Affect; CD4 Positive T Lymphocytes; CRISPR screen; Cell Fraction; Cell Line; Cells; Code; Complex; Development; EZH2 gene; Gene Expression; Genes; Goals; HIV; HIV Infections; HIV/AIDS; Health; Immunoprecipitation; Individual; Investigation; Knock-out; Laboratories; Libraries; MALAT1 gene; Mission; Mutate; National Institute of Allergy and Infectious Disease; Outcome; PVT1 gene; Phenotype; Polycomb; Population; Proteins; Public Health; RNA; RNA Splice Sites; Regulation; Regulator Genes; Reporter; Repression; Research; Rest; Role; Specificity; TNF gene; Testing; Therapeutic Intervention; Tissues; Toxic effect; Transcription Repressor; United States National Institutes of Health; Untranslated RNA; Viral; antiretroviral therapy; burden of illness; candidate identification; cell type; chromatin immunoprecipitation; combat; crosslink; fighting; knock-down; latent HIV reservoir; novel; promoter; side effect; small molecule; therapeutic RNA

PROJECT SUMMARY / ABSTRACT Establishment of the long-lasting latent reservoir and the low level of detectable HIV replication remain stumbling blocks to cure from HIV infection. Long non-coding RNAs (lncRNAs), novel regulators of gene expression, hold promise for development of HIV cure strategies, because they are more tissue and cell-type specific than protein coding genes. Our long-term goal is to develop more potent strategies with higher specificity than are currently available to target the latent HIV reservoir for reactivation or silencing. The overall objective of this application is to evaluate the merit of using lncRNA-based approaches for HIV cure. Our central hypothesis is that targeting single lncRNAs or their combinations will result in more robust effects on HIV expression compared to existing strategies that use latency reversing (LRAs) or latency promoting agents (LPAs). To test our central hypothesis, two specific aims will be pursued: (1) Identify lncRNAs with a role in the regulation of HIV expression by conducting a CRISPR/Cas9 screen; (2) Identify overlapping functions of lncRNAs using the RNA interactome of the HIV repressor zeste 2 polycomb repressive complex 2 subunit (EZH2). For the CRISPR/Cas9 screen, JLat cell lines constitutively expressing Cas9 will be generated and transduced with the sgRNA library to target lncRNA splice sites. To identify lncRNAs that act as HIV repressors, cells will be screened in the resting state. When repressive lncRNAs are knocked out, sgRNAs are expected to be enriched in the population of HIV-producing cells that express GFP reporter protein. To identify lncRNAs that act as HIV activators, the fraction of cells that remain GFP-negative after initial lncRNA knockout will be treated with tumor necrosis factor (TNF) alpha. When lncRNAs that contribute to HIV activation are knocked out, sgRNAs are expected to be enriched in the population of cells that remain GFP-negative following treatment with TNF alpha. For identification of overlapping functions of lncRNAs, EZH2 has been selected because it is an HIV repressor that has several known lncRNA interacting partners. We will identify the entire lncRNA interactome of EZH2 using the enhanced UV cross-linking and immunoprecipitation (eCLIP). Selected lncRNAs from the screen and the EZH2 interactome will be knocked down individually and in combinations in JLat and primary CD4+ T cells, and effect on HIV expression will be compared to that induced by LRAs and LPAs. We will further conduct chromatin immunoprecipitation to determine whether knockdown of identified lncRNAs affects EZH2 occupancy of the HIV promoter. Because eCLIP will result in identification of specific sequences of the lncRNAs that interact with EZH2, we will mutate these sequences to determine whether interaction with EZH2 is responsible for the observed activator or repressor phenotype. These results will be significant because they will provide strong scientific justification for development of lncRNA-based therapeutic interventions to reactivate latent HIV or enhance viral suppression on antiretroviral therapy. For example, small molecules can be used to specifically disrupt interactions between lncRNAs and proteins.

项目摘要/摘要 长期潜伏蓄水池的建立和可检测到的低水平艾滋病毒复制仍然存在 治愈艾滋病毒感染的绊脚石。长非编码RNA(LncRNAs)--新的基因调控因子 表达，为艾滋病毒治疗策略的发展提供了希望，因为它们更多的是组织和细胞类型 比蛋白质编码基因更具特异性。我们的长期目标是开发更强大的战略和更高的 特异性比目前可用于靶向重新激活或沉默的潜在艾滋病毒储存库的特异性更高。整体而言 这项应用的目的是评估使用基于lncRNA的方法治疗HIV的优点。我们的 中心假设是，靶向单个lncRNA或其组合将导致更强大的影响 HIV表达与使用潜伏期逆转(LRA)或潜伏期促进剂的现有策略的比较 (LPA)。为了验证我们的中心假设，我们将追求两个具体的目标：(1)确定在 通过进行CRISPR/Cas9筛查来调节HIV的表达；(2)确定重叠的功能 利用HIV抑制子Zest2多梳抑制复合体2亚单位的RNA相互作用组的LncRNAs (EZH2)。对于CRISPR/Cas9筛选，将产生组成性表达Cas9的JLat细胞系，并 用sgRNA文库转导靶向LncRNA剪接位点。识别起HIV作用的lncRNA 抑制物，细胞将在静息状态下进行筛选。当抑制性的lncRNA被敲除时，sgRNA被 预计将在表达GFP报告蛋白的艾滋病毒产生细胞群中得到丰富。要确定 作为HIV激活剂的lncRNA，在最初的lncRNA敲除后保持GFP阴性的细胞比例 将接受肿瘤坏死因子(肿瘤坏死因子)α治疗。当有助于HIV激活的lncRNAs 被敲除后，sgRNAs有望在GFP阴性的细胞群中得到丰富 在使用肿瘤坏死因子α治疗后。为了鉴定lncRNAs的重叠功能，EZH2已经被 之所以选择它，是因为它是一种艾滋病毒抑制因子，具有几个已知的lncRNA相互作用伙伴。我们将确定 利用增强型紫外光交联和免疫沉淀(ECLIP)获得了EZH2的整个lncRNA相互作用体。 从屏幕上选择的lncRNA和EZH2互动组将被单独和在 在JLat和原代CD4+T细胞中的组合以及对HIV表达的影响将与诱导的 由LRA和LPA提供。我们将进一步进行染色质免疫沉淀，以确定是否击倒 已识别的lncRNAs影响HIV启动子的EZH2占位。因为eCLIP将导致识别 与EZH2相互作用的lncRNAs的特定序列，我们将突变这些序列来确定 与EZH2的相互作用是否导致了观察到的激活子或抑制子表型。这些结果 将具有重要意义，因为它们将为基于lncRNA的开发提供强有力的科学依据 重新激活潜伏的艾滋病毒或加强抗逆转录病毒治疗的病毒抑制的治疗干预。为 例如，小分子可以专门用来破坏lncRNAs和蛋白质之间的相互作用。