Sorting and Sequencing Latent Reservoirs in HIV+ Opioid Users

摘要 阿片类药物使用者是艾滋病毒治疗战略的主要受益者，因为他们的医疗和社会脆弱性以及 对抗逆转录病毒治疗的依从性低，但使用吗啡或海洛因的人的潜伏库的性质 是未知的。这一多PI方案的中心假设是来自阿片类药物使用者的HIV DNA+细胞表现出独特的 转录和蛋白质组签名，提供对启动、建立和 维护吸毒者的潜伏蓄水池。这一假设是基于我们最近在《自然》杂志上的数据 使用一种新的分类和测序策略在无需预先激活的情况下表征HIV DNA+CD4+T细胞 查找-序列我们进一步表明，在潜伏感染的细胞中，特定的沉默和细胞生存途径发生了变化。 并确定了55个差异表达基因(DEG)，这些基因与HIV潜伏的潜在生物学有关。这里 我们建议将这些研究扩展到阿片类药物使用者的组织储存库，测试DEGS的功能相关性 在潜伏期生物学上，并发展重要的基因组、蛋白质组和生化进展的Find-Seq。 中心假设将在四个具体目标中得到检验：1)确定艾滋病毒持续存在的细胞机制 以及吗啡对感染艾滋病毒的T细胞和最后一次礼物参与者的小胶质细胞的调节作用。我们将使用 FIND-SEQ从最后一次送礼参与者的脾、肠道和脑中分离HIV+细胞并对其进行RNA测序 在没有吗啡的情况下，阐明阿片类药物对潜伏感染的T细胞和不同组织中的小胶质细胞的作用。 2)确定DEGS和阿片类药物如何调节T细胞和小胶质细胞中的HIV潜伏期。我们将在体外进行 分离的CD4+T细胞和小胶质细胞的CRISPR激活/干扰实验 多能干细胞以确定已鉴定的DEG与潜伏期建立的功能相关性 不含吗啡或海洛因。3)从小鼠脾组织中提取HIV+CD4+T细胞进行蛋白质组学分析 礼物参与者。我们将使用dab-seq，它允许高覆盖率的基因组测序和同步 最后一次送礼受试者脾细胞表面蛋白表达的分析 吗啡以确定前病毒的完整性、整合位置和蛋白表达的变化 阿片类药物使用引起的潜伏感染细胞。4)同时对RNA、蛋白质和HIV全基因组进行测序 来自HIV DNA+细胞。我们将把Find-Seq开发成一种多模式分析，在流中对HIV DNA+细胞进行分类 同时对回收的细胞进行基因组、转录和蛋白质组学分析。我们 组建了一支由艾滋病毒生物学和高级研究领域的世界专家组成的高度互补的团队 与尖端微流控、CRISPR和多组学测序技术相结合的延迟 来自Last Gift Cohort和Johns的艾滋病毒携带者和阿片类药物使用患者临床护理的领先专家 霍普金斯艾滋病临床队列。我们期待着获得改变范式的洞察力，深入了解“潜在的分子 HIV潜伏期在中枢神经系统、淋巴系和髓系中启动、建立和维持的机制 以及物质使用可能如何影响这些过程“，并与本RFA完全一致。

ABSTRACT Opioid users are prime beneficiaries of cure strategies for HIV due to their medical and social vulnerabilities and low adherence to antiretroviral therapies, but the nature of the latent reservoir in people using morphine or heroin is unknown. The central hypothesis of this multi-PI proposal is that HIV DNA+ cells from opioid users show unique transcriptional and proteomic signatures that provide fundamental insight into initiation, establishment and maintenance of the latent reservoir in drug users. This hypothesis is based on our recent data in Nature characterizing HIV DNA+ CD4+ T cells without prior activation using a new sorting and sequencing strategy called FIND-Seq. We further show that specific silencing and cell survival pathways are altered in latently infected cells and identify 55 differentially expressed genes (DEGs) implicated in the underlying biology of HIV latency. Here we propose to extend these studies to tissue reservoirs from opioid users, test the functional relevance of DEGs on latency biology, and develop important genomic, proteomic and biochemical advancements of FIND-Seq. The central hypothesis will be tested in four specific aims: 1) Define the cellular mechanisms of HIV persistence and their modulation by morphine in HIV-infected T cells and microglia from Last Gift participants. We will use FIND-seq to sort and RNA sequence HIV+ cells from the spleen, gut, and brain of Last Gift participants with and without morphine to elucidate the role of opioids on latently infected T cells and microglia across different tissues. 2) Determine how DEGs and opioids regulate HIV latency in T cells and microglia. We will perform ex vivo CRISPR activation/interference experiments with isolated CD4+ T cells and microglia derived from induced pluripotent stem cells to determine the functional relevance of identified DEGs for latency establishment with and without morphine or heroin. 3) Perform a proteogenomic analysis of HIV+CD4+ T cells from the spleen of Last Gift participants. We will use DAb-seq, which allows high-coverage genome sequencing and simultaneous analysis of cell-surface protein expression on CD4+ T cells from spleens from Last Gift participants with or without morphine to determine the intactness of the provirus, its integration site, and changes in protein expression of latently infected cells induced by opioid use. 4) Simultaneously sequence the RNA, protein, and full HIV genome from HIV DNA+ cells. We will develop FIND-Seq into a multimodal assay that sorts HIV DNA+ cells on a flow cytometer and simultaneously performs genomic, transcriptomic, and proteomic analysis on recovered cells. We have assembled a highly complementary team of world experts in the biology and advanced investigation of HIV latency with cutting-edge microfluidics, CRISPR and multi-omics sequencing technologies combined with leading experts in clinical care of people living with HIV and opioid use from the Last Gift Cohort and the Johns Hopkins HIV Clinical Cohort. We expect to gain paradigm-shifting insight into the “underlying molecular mechanisms by which HIV latency is initiated, established, and maintained in the CNS, lymphoid and myeloid tissues and how substance use might influence these processes” and are fully aligned with this RFA.