Understanding HIV reservoir formation by profiling transcriptomic and epigenetic changes in CD4 T cells following ART initiation

在艾滋病毒感染期间，潜伏感染细胞的储存库在治疗期间持续存在，并且 这个蓄水池是治愈艾滋病毒的主要障碍。我们和其他人已经证明，艾滋病毒 潜伏期是一种表观遗传现象，其特征是染色质结构的明显变化 以及抑制HIV基因表达的组蛋白修饰，这些基因可以通过细胞传播 组织。潜伏期的这种可遗传特性允许潜伏的表型在正在进行的克隆中存活下来 宿主中受感染细胞的扩张，并维持整个储存库。此外，病毒沉默 表观遗传编程与CD4T细胞内发生的全球变化有关，因为它们 从激活状态转换到休眠状态。充分定义储油层形成的过程 形成并编程延迟将是指导新方法的关键 防止水库播种或维护。到目前为止，研究成藏过程 在临床上，艾滋病毒感染被认为是困难的，如果不是不可能的，因为以前 观察表明，水库在急性感染期间很早就形成了。更新的 然而，来自几个组织的信息现在显示，大多数水库是 在ART启动时被活跃复制的病毒播种，这表明ART 启动触发了水库的播种。这一观察结果提出了令人兴奋的可能性 我们可以通过对CD4T的纵向观察来研究水库播种的过程 通过这样做，揭示了潜在的方法来 在治疗开始时阻止储存层的形成。在这项提案中，我们的目标是 全面定义在CD4T细胞中发生的表观遗传学和基于染色质的变化 使用来自A5248队列的样本进行抗逆转录病毒治疗。这些样本中的CD4T细胞 将使用一系列尖端分析方法进行分析，这些分析方法已在 我们的实验室。这些检测包括联合单细胞多组体(RNAseq/ATACseq) 通过以下途径对多个关键的组蛋白修饰进行分析和高分辨率全基因组作图 在靶子下切割并使用核酸酶释放(切割和运行)。通过这种方法，我们将 实现对基于染色质的可遗传模式如何改变的综合理解 CD4T细胞由ART启动触发，并产生对 与体内水库播种相吻合的细胞内条件。通过实现这一目标，我们将 揭示促进油藏播种的机制并提出新的封堵方法 在ART启动时形成的油藏。

During HIV infection, a reservoir of latently infected cells forms that persists during therapy, and this reservoir represents the major barrier to an HIV cure. We and others have shown that HIV latency is an epigenetic phenomenon, characterized by distinct changes to chromatin structure and histone modifications that repress HIV gene expression, that can be transmitted through cell division. This heritable property of latency allows the latent phenotype to survive ongoing clonal expansion of infected cells in hosts and sustain the overall reservoir. Furthermore, viral silencing and epigenetic programming is linked to global changes that occur within CD4 T cells as they transition from an activated to a resting state. Fully defining the process by which the reservoir is formed and by which latency is programmed will be essential to guiding novel approaches to prevent reservoir seeding or maintenance. To date, studying the process of reservoir formation during clinical HIV infection has been considered difficult, if not impossible, due to prior observations suggesting that the reservoir forms very early during acute infection. Newer information from several groups, however, has now revealed that the majority of the reservoir is seeded by viruses that are actively replicating at the time of ART initiation, suggesting that ART initiation triggers seeding of the reservoir. This observation raises the exciting possibility that we could potentially study the process of reservoir seeding by longitudinal observation of CD4 T cells from PWH in the immediately post ART period and, by doing so, reveal potential ways to block the reservoir from forming at the time of therapy initiation. In this proposal we aim to comprehensively define epigenetic and chromatin-based changes that occur in CD4 T cells in the post ART period using samples derived from the A5248 cohort. CD4 T cells from these samples will be profiled using a series of cutting-edge assays that have been established and validated in our laboratory. These assays include combined single-cell multi-omic (RNAseq/ATACseq) analysis, and high-resolution genome-wide mapping of multiple key histone modifications through Cleavage Under Targets and Release using Nuclease (CUT&RUN). From this approach we will achieve an integrated understanding of how heritable patterns of chromatin-based changes in CD4 T cells are triggered by ART initiation and generate novel and critical insights into the intracellular conditions that coincide with reservoir seeding in vivo. By achieving this goal, we will reveal mechanisms that promote reservoir seeding and suggest novel approaches to block reservoir formation at the time of ART initiation.