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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要长期潜伏病毒库的建立和可检测到的 HIV 复制水平仍然较低治愈艾滋病毒感染的绊脚石。长链非编码RNA（lncRNA），新型基因调控因子表达，有望开发艾滋病毒治疗策略，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体。我们的长期目标是制定更有效、更高水平的战略比目前针对潜在 HIV 病毒库进行重新激活或沉默的特异性更高。整体该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，针对单个 lncRNA 或其组合将导致更强大的效应 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）。为了检验我们的中心假设，我们将追求两个具体目标：（1）识别在通过 CRISPR/Cas9 筛选调节 HIV 表达； (2) 识别重叠的职能使用 HIV 抑制因子 zeste 2 多梳抑制复合物 2 亚基的 RNA 相互作用组的 lncRNA （EZH2）。对于 CRISPR/Cas9 筛选，将生成组成型表达 Cas9 的 JLat 细胞系并用 sgRNA 文库转导至目标 lncRNA 剪接位点。识别充当 HIV 的 lncRNA 阻遏物，细胞将在静息状态下进行筛选。当抑制性lncRNA被敲除时，sgRNA 预计在表达 GFP 报告蛋白的 HIV 产生细胞群中富集。识别作为 HIV 激活剂的 lncRNA，初始 lncRNA 敲除后仍保持 GFP 阴性的细胞比例将接受肿瘤坏死因子 (TNF) α 治疗。当有助于 HIV 激活的 lncRNA 被 sgRNA 被敲除后，预计将在 GFP 阴性的细胞群中富集用 TNF α 治疗后。为了识别 lncRNA 的重叠功能，EZH2 已被之所以选择它，是因为它是一种 HIV 抑制因子，具有多个已知的 lncRNA 相互作用伙伴。我们将确定使用增强型紫外交联和免疫沉淀 (eCLIP) 分析 EZH2 的整个 lncRNA 相互作用组。从屏幕中选择的 lncRNA 和 EZH2 相互作用组将被单独敲除 JLat 和原代 CD4+ T 细胞中的组合，并对 HIV 表达的影响将与诱导的效果进行比较由 LRA 和 LPA 执行。我们将进一步进行染色质免疫沉淀以确定是否敲低已鉴定的 lncRNA 影响 HIV 启动子的 EZH2 占据。因为 eCLIP 将导致识别与 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 的相互作用是否是观察到的激活子或阻遏子表型的原因。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增强抗逆转录病毒治疗的病毒抑制的治疗干预措施。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。