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.

项目总结/摘要 建立长期潜伏的水库和可检测到的艾滋病毒复制水平低的情况仍然存在 是治愈艾滋病的绊脚石。长链非编码RNA（lncRNA），基因的新调控因子 表达，为开发HIV治疗策略带来希望，因为它们是更多的组织和细胞类型 比蛋白质编码基因更特异。我们的长期目标是开发更有效的策略， 这比目前可用于靶向潜伏的HIV储库进行再激活或沉默的特异性更强。整体 本申请的目的是评价使用基于lncRNA的方法治疗HIV的优点。我们 中心假设是靶向单个lncRNA或其组合将导致对细胞的更强的作用。 与使用潜伏期逆转（LRA）或潜伏期促进剂的现有策略相比， （LPAs）。为了检验我们的中心假设，将追求两个具体目标：（1）鉴定在细胞凋亡中起作用的lncRNA。 通过进行CRISPR/Cas9筛选来调节HIV表达;（2）确定 使用HIV阻遏物zeste 2多梳阻遏复合物2亚基的RNA相互作用组的lncRNA （EZH2）。对于CRISPR/Cas9筛选，将产生组成型表达Cas9的JLat细胞系，并将其转化为细胞。 用sgRNA文库转导以靶向lncRNA剪接位点。为了鉴定出作为艾滋病病毒的lncRNA 抑制子，将在静息状态下筛选细胞。当抑制性lncRNA被敲除时， 预期在表达GFP报告蛋白的HIV产生细胞群体中富集。以识别 作为HIV激活剂的lncRNA，在初始lncRNA敲除后保持GFP阴性的细胞部分 将接受肿瘤坏死因子（TNF）α治疗。当参与HIV激活的lncRNA被 敲除后，预期sgRNA在保持GFP阴性的细胞群体中富集 用TNF α治疗后。为了鉴定lncRNA的重叠功能，EZH 2已经被鉴定。 选择它是因为它是一种HIV阻遏物，具有几种已知的lncRNA相互作用伴侣。我们将确定 使用增强的紫外线交联和免疫沉淀（eCLIP）来研究EZH 2的整个lncRNA相互作用组。 来自筛选和EZH 2相互作用组的选择的lncRNA将被单独敲除，并且与EZH 2相互作用组的lncRNA相互作用。 在JLat和原代CD 4 + T细胞中的组合，并且将对HIV表达的作用与诱导的作用进行比较。 LRA和LPA。我们将进一步进行染色质免疫沉淀，以确定是否敲除 鉴定的lncRNA影响HIV启动子的EZH 2占据。因为eCLIP将导致识别 与EZH 2相互作用的lncRNA的特定序列，我们将突变这些序列以确定 与EZH 2的相互作用是否负责观察到的激活子或阻遏子表型。这些结果 将是重要的，因为它们将为开发基于lncRNA的药物提供强有力的科学依据。 治疗干预措施，以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。为 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。