Multi-omic dissection of the transcriptional, epigenetic, and proteomic signatures of cells infected with latent HIV

项目总结 一小部分长期存活的CD4T细胞携带有复制能力的病毒(潜伏的艾滋病毒宿主) 有效的抗逆转录病毒治疗，即使在病毒血症无法检测到的情况下；这种潜伏的艾滋病毒宿主总是 与停止治疗时病毒反弹有关。潜伏的艾滋病毒宿主是治愈艾滋病的主要障碍 艾滋病毒，但多重技术挑战限制了其调查。我们开发了一种超高通量液滴 被称为聚合酶链式反应激活的细胞分选(PACS)的微流控工作流程，用于检测、分类和测序单个细胞 含有细胞内HIV DNA的单一拷贝。在初步研究中，我们展示了PACS的能力 为了1)以超高吞吐量处理数百万个细胞，2)检测感染艾滋病毒的细胞3)单个细胞分类和排序 这群罕见的潜伏感染的CD4T细胞。基于这些发现，我们认为PACS提供了一种 克服现有技术挑战并首次定义基因组的独特机会 控制艾滋病毒潜伏储存库的机制。我们假设潜伏感染的细胞具有独特的 使它们能够在不产生病毒的情况下拥有复制能力的艾滋病毒基因组的特性。因此， 我们建议将PACS与单细胞RNA-SEQ、基因组和整合位点分析方法相结合， ATAC-SEQ和蛋白质组学，以确定控制单个HIV潜伏库的基因组机制 受感染的细胞来自ART抑制病毒的人。我们的具体目标如下： 具体目标1：确定携带潜伏病毒的单细胞的转录组。我们建议按顺序排列 从个体血液中分离的单个HIV阳性的CD4T细胞的转录本，而 平行测定HIV前病毒序列和插入位点。这些研究将定义转录 携带全长复制能力病毒的潜伏感染细胞的程序。 特异性目标2：建立携带潜伏病毒的单细胞染色质图谱。我们建议 分析单个HIV阳性CD4T细胞的染色质图谱，以确定宿主之间的关系 DNA染色质状态和HIV潜伏期。这些研究将确定染色质状态在控制中的作用 并确定潜伏感染的CD4T细胞转录程序的调节因子。 具体目标3：确定潜伏感染的CD4T细胞的表面标志。我们建议潜伏分离HIV病毒 用PACS感染CD4T细胞，并用条形码抗体测序分析其表面蛋白质组 确定最能确定HIV潜伏库的表面标记组合。完成后，这些研究将 将定义新的表面标记组合来识别潜伏感染的CD4T细胞。 总而言之，这些研究使用了基于我们在基因组方面的综合专业知识的多组学方法 免疫细胞、艾滋病毒生物学和微流体的调节，以确定控制艾滋病毒潜伏期的机制。 因此，这些研究将指导新的治疗干预措施的发展，同时提供新的工具 用于监测接受抗逆转录病毒治疗的感染者中潜在的艾滋病毒蓄积物。

PROJECT SUMMARY A small population of long-lived CD4 T cells harbors replication competent virus (the latent HIV reservoir) during effective antiretroviral therapy even when viremia is undetectable; this latent HIV reservoir is invariably associated with virus rebound when treatment is stopped. The latent HIV reservoir is a major barrier to curing HIV, but multiple technical challenges limit its investigation. We developed an ultra-high throughput droplet microfluidic workflow called PCR activated cell sorting (PACS) that detects, sorts and sequences single cells containing a single copy of intracellular HIV DNA. In preliminary studies we demonstrated the ability of PACS to 1) process millions of cells at ultra-high throughput, 2) detect HIV infected cells 3) single cell sort and sequence this rare population of latently infected CD4 T cells. Based on these findings, we believe that PACS provides a unique opportunity to overcome existing technical challenges and define, for the first time, the genomic mechanisms that control the HIV latent reservoir. We hypothesize that latently infected cells have unique properties that allow them to harbor replication competent HIV genomes without producing virus. Thus, we propose to combine PACS with methods for single cell RNA-seq, genome and integration site analysis, ATAC-seq and proteomics to define the genomic mechanisms that control the HIV latent reservoir in single infected cells from people with ART suppression of the virus. Our Specific Aims are as follows: Specific Aim 1: Define the transcriptome of single cells harboring latent virus. We propose to sequence the transcriptomes of single HIV positive CD4 T cells isolated from the blood of individuals on ART, while determining in parallel the HIV provirus sequence and insertion site. These studies will define the transcriptional program of latently infected cells harboring full-length replication competent virus. Specific Aim 2: Establish the chromatin landscape of single cells harboring latent virus. We propose to analyze the chromatin profile of single HIV positive CD4 T cells in order to define the relationship between host DNA chromatin status and HIV latency. These studies will determine the role of chromatin status in the control of HIV latency and identify regulators of the transcriptional program of latently infected CD4 T cells. Specific Aim 3: Identify surface markers of latently infected CD4 T cells. We propose to isolate HIV latently infected CD4 T cells using PACS and analyze their surface proteome using barcoded antibody sequencing to identify surface marker combinations that best define the HIV latent reservoir. Upon completion, these studies will define novel surface marker combinations to identify latently infected CD4 T cells. IN SUMMARY, these studies use a multi-omics approach based on our combined expertise in the genomic regulation of immune cells, HIV biology, and microfluidics to define the mechanisms that control HIV latency. Hence, these studies will guide the development of novel therapeutic interventions, while providing novel tools for the monitoring of the latent HIV reservoir in infected individuals undergoing ART.