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: 10402718
• 负责人: Nadejda S Beliakova-Bethell
• 金额: $ 21.53万
• 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/10402718
• 关键词: AIDS/HIV problem; Affect; CD4 Positive T Lymphocytes; CRISPR screen; Cell Fraction; Cell Line; Cells; Code; Complex; Development; EZH2 gene; Gene Expression; Genes; Goals; HIV; HIV Infections; 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; base; 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 建立持久的潜在水库和低水平可检测的HIV复制水平仍然存在 绊脚石可从HIV感染中治愈。长非编码RNA（LNCRNA），基因的新调节剂 表达，保持艾滋病毒治疗策略的发展，因为它们是更多的组织和细胞类型 特异性比蛋白质编码基因。我们的长期目标是制定更有效的策略 特异性比目前可用于针对潜在的艾滋病毒储存库进行重新激活或沉默的特异性。总体 该应用的目的是评估使用基于LNCRNA的方法进行HIV治疗的优点。我们的 中心假设是靶向单个LNCRNA或其组合将对 与使用潜伏期逆转（LRA）或促进剂的现有策略相比，艾滋病毒表达 （LPA）。为了检验我们的中心假设，将追求两个具体目标：（1）确定在 通过进行CRISPR/CAS9屏幕来调节HIV表达； （2）确定重叠功能 LNCRNA使用HIV阻遏物Zeste 2 Polycomb抑制剂复合物2亚基的RNA相互作用组 （EZH2）。对于CRISPR/CAS9屏幕，将生成组成性表达Cas9的JLAT细胞系，并且 用SGRNA文库转导向靶向LNCRNA剪接位点。识别充当艾滋病毒的lncRNA 阻遏物，细胞将以静止状态进行筛选。当抑制性lncrnas被淘汰时，sgrnas是 预计将富含表达GFP报告蛋白的HIV产生细胞的种群。识别 充当HIV激活剂的LNCRNA，初始lncRNA敲除后保持GFP阴性的细胞的比例 将用肿瘤坏死因子（TNF）α进行治疗。当有助于HIV激活的LNCRNA是 被淘汰，预计SGRNA将富集在保持GFP阴性的细胞群中 用TNF alpha处理后。为了识别LNCRNA的重叠函数，EZH2已经 之所以选择是因为它是一种HIV阻遏物，具有几个已知的LNCRNA相互作用伙伴。我们将确定 使用增强的紫外线交联和免疫沉淀（Eclip）的EZH2的整个LNCRNA相互作用。 从屏幕和EZH2 Interactome中选定的lncRNA将被单独击倒 将JLAT和主要CD4+ T细胞组合，以及对HIV表达的影响与诱导的影响 由LRA和LPA。我们将进一步进行染色质免疫沉淀，以确定是否敲低 确定的LNCRNA会影响HIV启动子的EZH2占用率。因为Eclip将导致确定 与EZH2相互作用的LNCRNA的特定序列，我们将突变这些序列以确定 与EZH2的相互作用是导致观察到的激活剂还是阻遏物表型。这些结果 之所以重要，是因为它们将为基于LNCRNA的发展提供强有力的科学理由 治疗干预措施可重新激活潜在的HIV或增强抗逆转录病毒治疗的病毒抑制。为了 例如，小分子可用于特异性破坏LNCRNA和蛋白质之间的相互作用。