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

对潜伏 HIV 感染细胞的转录、表观遗传和蛋白质组学特征进行多组学分析

• 批准号: 10447107
• 负责人: Adam R. Abate
• 金额: $ 75.03万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10447107
• 关键词: ATAC-seq; Address; Antibodies; Bar Codes; Biological Markers; Biology; Blood; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Catalogs; Cell Separation; Cells; Chromatin; Custom; DNA; Data; Defective Viruses; Detection; Development; Dissection; Epigenetic Process; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome; Genomic DNA; Genomics; Goals; HIV; HIV Genome; HIV Infections; HIV Seropositivity; In Vitro; Individual; Infection; Investigation; Length; Life Cycle Stages; Link; Membrane Proteins; Messenger RNA; Methods; Microfluidics; Monitor; Natural Killer Cells; Pathway interactions; Persons; Physiological; Population; Process; Property; Proteins; Proteome; Proteomics; Proviruses; Regulation; Research; Role; Sampling; Signal Transduction; Site; Surface; System; T-Lymphocyte; Testing; Therapeutic; Time; Transcriptional Regulation; Transposase; Viral; Viral Markers; Viremia; Virus; Virus Latency; antiretroviral therapy; base; digital; genome-wide analysis; high throughput analysis; immunoregulation; integration site; interest; latent HIV reservoir; multiple omics; novel; novel strategies; novel therapeutic intervention; novel therapeutics; programs; proteomic signature; single cell sequencing; single-cell RNA sequencing; tool; transcription factor; transcriptome; transcriptome sequencing; viral rebound

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.

项目概要 一小群长寿的 CD4 T 细胞在复制过程中携带有复制能力的病毒（潜在的 HIV 病毒库）。 即使无法检测到病毒血症，也能进行有效的抗逆转录病毒治疗；这种潜在的艾滋病毒储存库总是 与治疗停止时病毒反弹有关。潜在的艾滋病毒储存库是治愈的主要障碍 HIV，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 称为 PCR 激活细胞分选 (PACS) 的微流体工作流程，可检测、分选和测序单细胞 含有单拷贝的细胞内HIV DNA。在初步研究中，我们展示了 PACS 的能力 1) 以超高通量处理数百万个细胞，2) 检测 HIV 感染细胞 3) 单细胞分选和测序 这种罕见的潜伏感染 CD4 T 细胞群。基于这些发现，我们相信 PACS 提供了 克服现有技术挑战并首次定义基因组的独特机会 控制HIV潜伏病毒库的机制。我们假设潜伏感染的细胞具有独特的 这些特性使它们能够携带具有复制能力的艾滋病毒基因组而不产生病毒。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 和蛋白质组学定义了控制 HIV 潜伏病毒库的基因组机制 来自接受病毒 ART 抑制的人的感染细胞。我们的具体目标如下： 具体目标 1：定义含有潜伏病毒的单细胞的转录组。我们建议排序 从 ART 个体的血液中分离出单个 HIV 阳性 CD4 T 细胞的转录组，同时 并行确定 HIV 原病毒序列和插入位点。这些研究将定义转录 含有全长复制能力病毒的潜伏感染细胞程序。 具体目标 2：建立含有潜伏病毒的单细胞染色质景观。我们建议 分析单个 HIV 阳性 CD4 T 细胞的染色质谱，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对照中的作用 HIV 潜伏期并确定潜伏感染 CD4 T 细胞转录程序的调节因子。 具体目标 3：识别潜伏感染 CD4 T 细胞的表面标记。我们建议隔离HIV潜伏者 使用 PACS 感染 CD4 T 细胞，并使用条形码抗体测序分析其表面蛋白质组 确定最能定义 HIV 潜伏病毒库的表面标记组合。这些研究完成后 将定义新的表面标记组合来识别潜伏感染的 CD4 T 细胞。 总之，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、HIV 生物学和微流体的调节，以确定控制 HIV 潜伏期的机制。 因此，这些研究将指导新型治疗干预措施的开发，同时提供新的工具 用于监测接受 ART 的感染者的潜在 HIV 储存库。