The role of RNA m6A modification in the regulation of HIV latency and reactivation

RNA m6A 修饰在调节 HIV 潜伏和再激活中的作用

• 批准号: 10600078
• 负责人: JONATHAN KARN
• 金额: $ 71.55万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10600078
• 关键词: Adenosine; Antibodies; Behavior; Biological Assay; Cell model; Cells; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Defect; Epigenetic Process; Event; Gene Expression; Genes; Genetic Transcription; Goals; HIV; HIV Infections; HIV tat Protein; Immunoprecipitation; Informatin; Interleukin-15; Jurkat Cells; Kinetics; Knock-out; Laboratories; Maps; Measures; Meclofenamic Acid; Messenger RNA; Metabolic; Methylation; Modeling; Modification; Mutagenesis; Nature; Nuclear Export; Pathway interactions; Patients; Phase; Phenotype; Play; Post-Transcriptional RNA Processing; Post-Transcriptional Regulation; Proviruses; RNA; RNA Splicing; RNA Stability; RNA methylation; Reader; Regimen; Regulation; Resolution; Role; Sampling; Shock; Site; System; T memory cell; T-Lymphocyte; Technology; Testing; Therapeutic; Transcript; Transportation; Up-Regulation; Virion; Virus; Virus Latency; Virus Replication; Visualization; Work; acute infection; cell transformation; cellular imaging; design; epitranscriptomics; experience; experimental study; histone demethylase; in vivo; inhibitor; knock-down; mRNA Precursor; multidisciplinary; nanopore; next generation sequencing; pharmacologic; posttranscriptional; protein expression; reactivation from latency; response; single-cell RNA sequencing; small hairpin RNA; synergism; transcriptome sequencing; viral RNA

Background. This proposal, which is submitted in response to RFA-AI-21-021 “Understanding Post-Transcriptional Regulation of Intact and Defective HIV RNA”. N6-methyladenosine (m6A), is the most common RNA modification and is known to regulate RNA stability, splicing and nuclear export. m6A modification of HIV transcripts is crucial for the early stages of HIV infection during acute infection of primary T cells, but it is an open question whether m6A modification controls HIV latency and reactivation in ART-suppressed patients. Our goal. Our multidisciplinary team has extensive experience in studies of HIV latency and reactivation in patients and in reliable primary cell models, studies of RNA m6A modification, and cutting-edge technologies such as NGS sequencing and scRNA-seq analysis. To overcome the challenge of measuring m6A in RNA recovered from the extremely low numbers of HIV+ cells present in patient samples, we will develop a sensitive next-generation sequencing assay for the profiling and quantification of m6A modification in different HIV transcripts from patient samples. This assay, which we call MeRIP-EDITS combines methylated RNA immunoprecipation with the EDITS assay, which has been used in multiple clinical studies to measure the inducible HIV reservoir. We will use the MeRIP-EDITS assay to characterize m6A modification of different HIV transcripts at different reactivation kinetic points of latent HIV and examine changes of the m6A pathway during HIV latency and reactivation. In parallel we will perform mechanistic studies on the m6A pathway using the QUECEL primary cell model of HIV latency. We will use the model to develop a sensitive nanopore RNA-sequencing assay which can subsequently be applied to patient samples. We will also inhibit the activity of the m6A writer METTL3 and the erasers FTO and ALKBH5 by knocking out the expression of these genes by using the CRISPR gene editing technology. High resolution mRNA FISH experiments, which distinguish between spliced and partially spliced HIV mRNA transcripts will be used to study the colocalization of m6A readers and HIV mRNAs. How will we advance the field? Demonstration of a central role of m6A in the control of HIV latency would immediately suggest pharmacological strategies to incorporate into HIV cure regimens. To date, it has been impossible to efficiently reverse HIV latency using agents that are designed for “kick and kill” strategies for an HIV cure. Using the sensitive assays described above, we will evaluate the impact of inhibitors of m6A erasers as part of a “kick and kill” strategy for HIV latency reversal. As a complementary approach we will also evaluate whether inhibitors of m6A writers can inhibit HIV reactivation and lead to long term silencing, as part of a “block and lock” strategy.

背景。该提案是为了回应 RFA-AI-21-021“理解 完整和缺陷 HIV RNA 的转录后调控”。N6-甲基腺苷 (m6A) 是最常见的 RNA 修饰，已知可调节 RNA 稳定性、剪接和 核出口。 HIV 转录本的 m6A 修饰对于 HIV 感染的早期阶段至关重要 在原代 T 细胞的急性感染期间，但 m6A 修饰是否会引起仍是一个悬而未决的问题 控制 ART 抑制患者的 HIV 潜伏期和重新激活。 我们的目标。我们的多学科团队在艾滋病毒潜伏期和研究方面拥有丰富的经验 在患者和可靠的原代细胞模型中重新激活，RNA m6A 修饰的研究，以及 NGS 测序和 scRNA-seq 分析等尖端技术。为了克服 测量从极少量 HIV+ 细胞中回收的 RNA 中 m6A 的挑战 存在于患者样本中，我们将开发一种灵敏的下一代测序测定法 对患者样本中不同 HIV 转录本中的 m6A 修饰进行分析和定量。 我们将这种检测方法称为 MeRIP-EDITS，将甲基化 RNA 免疫沉淀与 EDITS 测定，已用于多项临床研究来测量诱导型 HIV 水库。我们将使用 MeRIP-EDITS 测定来表征不同 HIV 的 m6A 修饰 在潜伏 HIV 的不同重新激活动力学点转录并检查 m6A 的变化 HIV潜伏期和重新激活期间的途径。与此同时，我们将进行机械研究 m6A 通路使用 HIV 潜伏期的 QUECEL 原代细胞模型。我们将使用该模型 开发一种灵敏的纳米孔 RNA 测序测定方法，随后可应用于 患者样本。我们还将抑制 m6A 写入器 METTL3 和擦除器 FTO 的活动 和 ALKBH5，通过使用 CRISPR 基因编辑敲除这些基因的表达 技术。高分辨率 mRNA FISH 实验，可区分剪接和剪接 部分剪接的 HIV mRNA 转录本将用于研究 m6A 阅读器的共定位和 HIV mRNA。 我们将如何推进该领域？证明 m6A 在控制 HIV 中的核心作用 潜伏期将立即提出将药物策略纳入艾滋病毒治疗的建议 治疗方案。迄今为止，使用以下药物还无法有效逆转 HIV 潜伏期： 专为治疗艾滋病毒的“踢杀”策略而设计。使用上述灵敏测定法， 我们将评估 m6A 擦除器抑制剂的影响，作为 HIV“踢杀”策略的一部分 潜伏期逆转。作为补充方法，我们还将评估 m6A 抑制剂是否 作家可以抑制艾滋病毒重新激活并导致长期沉默，作为“封锁和锁定”的一部分 战略。