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

背景本提案是根据RFA-AI-21-021“理解”提交的 完整和有缺陷的HIV RNA的转录后调节”。N6-甲基腺苷 (m6A)是最常见的RNA修饰，已知其调节RNA稳定性、剪接和 核出口。HIV转录本的m6 A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m6 A修饰是否是一个悬而未决的问题， 控制ART抑制患者的HIV潜伏期和再激活。 我们的目标。我们的多学科团队在HIV潜伏期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 m6 A修饰的研究，以及 NGS测序和scRNA-seq分析等尖端技术。克服 测量从极少量HIV+细胞中回收的RNA中m6 A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检测方法， 对来自患者样品的不同HIV转录物中的m6 A修饰进行分析和定量。 我们称之为MeRIP-EDITS的这种检测方法将甲基化RNA免疫沉淀与 EDITS检测，已用于多项临床研究，以测量诱导型HIV 水库我们将使用MeRIP-EDITS检测来表征不同HIV的m6 A修饰， 在潜伏期HIV的不同再活化动力学点的转录本，并检查m6 A的变化 HIV潜伏期和再激活期间的通路。与此同时，我们将进行机械研究， 使用HIV潜伏期的QUECEL原代细胞模型的m6 A途径。我们将使用该模型来 开发一种灵敏的纳米孔RNA测序测定法，随后可应用于 患者样本。我们还将抑制m6 A写入器胃L3和擦除器FTO的活性 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，其区分剪接和 部分剪接的HIV mRNA转录本将用于研究m6 A阅读器的共定位， HIV mRNA。 我们将如何推进这一领域？证明m6 A在控制艾滋病毒方面的核心作用 潜伏期将立即建议将药理学策略纳入艾滋病毒治疗 养生法到目前为止，不可能有效地逆转HIV潜伏期， 是为治疗艾滋病而设计的使用上述灵敏测定， 我们将评估m6 A橡皮擦抑制剂的影响，作为艾滋病毒“踢和杀”战略的一部分， 潜伏期逆转作为补充方法，我们还将评估m6 A抑制剂是否 作家可以抑制艾滋病毒的重新激活，并导致长期沉默，作为“封锁和锁定”的一部分， 战略

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.