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

背景资料。本提案是根据RFA-AI-21-021《谅解》提交的 完整和缺陷艾滋病毒RNA的转录后调控“。N6-甲基腺苷 (M6A)是最常见的RNA修饰，已知调节RNA的稳定性、剪接和 核出口。HIV转录本的M6A修饰对HIV感染的早期阶段至关重要 在急性感染原代T细胞期间，但m6A是否被修饰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 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。要克服这个问题 从极低数量的HIV+细胞中回收的RNA中检测m6A的挑战 在患者样本中，我们将开发一种灵敏的下一代测序分析方法，用于检测 患者样本中不同HIV转录本中m6A修饰的图谱和量化。 这种检测，我们称之为MERIP-EDITS，将甲基化的RNA免疫沉淀与 编辑试验，已被用于多项临床研究，以测量可诱导的艾滋病毒 水库。我们将使用MERIP-EDITS试验来表征不同HIV的m6A修饰 潜伏HIV在不同复活点的转录本及检测M6A的变化 在HIV潜伏期和重新激活期间的通路。同时，我们将对其进行机械研究 M6A通路采用QUECEL原代细胞模型的HIV潜伏期。我们将使用该模型来 建立一种灵敏的纳米孔RNA测序分析方法，可随后应用于 病人样本。我们还将抑制M6A写入器METTL3和擦除器FTO的活动 和ALKBH5通过CRISPR基因编辑敲除这些基因的表达 技术高分辨率的mRNA FISH实验，区分剪接和 部分剪接的HIV mRNA转录本将用于研究m6A阅读器和 HIV mRNAs。 我们将如何推进这一领域？证明M6A在艾滋病毒控制中的核心作用 潜伏期会立即建议将药物策略纳入艾滋病毒治疗 养生法。到目前为止，使用下列药物有效逆转艾滋病毒潜伏期是不可能的 专为艾滋病治愈的“踢杀”策略而设计。使用上述灵敏的分析方法， 我们将评估m6A橡皮擦抑制剂的影响，以此作为艾滋病病毒“先踢后杀”策略的一部分。 潜伏期反转。作为补充，我们还将评估M6A的抑制剂 写入者可以抑制艾滋病毒的重新激活，并导致长期沉默，这是“阻止和锁定”的一部分 策略。

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.