Sorting and Sequencing Latent Reservoirs in HIV+ Opioid Users

摘要 类阿片使用者由于其医疗和社会脆弱性，是艾滋病毒治疗战略的主要受益者， 对抗逆转录病毒疗法的依从性较低，但使用吗啡或海洛因的人的潜在储存库的性质 不明这个多PI提案的中心假设是，来自阿片类药物使用者的HIV DNA+细胞显示出独特的 转录和蛋白质组特征，提供了对启动，建立和 维持吸毒者的潜在储存库。这个假设是基于我们最近在《自然》杂志上的数据 在没有预先激活的情况下，使用称为 查找序列我们进一步表明，在潜伏感染的细胞中，特异性沉默和细胞存活途径发生了改变 并确定了55个差异表达基因（DEG），这些基因与HIV潜伏期的潜在生物学有关。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库，测试DEG的功能相关性 潜在生物学，并开发重要的基因组，蛋白质组学和生物化学进步的FIND-Seq。 中心假设将在四个具体目标进行测试：1）定义HIV持续存在的细胞机制 以及吗啡对“最后的礼物”参与者感染HIV的T细胞和小胶质细胞的调节作用。我们将使用 FIND-seq对来自最后一份礼物参与者的脾脏，肠道和大脑的HIV+细胞进行分类和RNA测序， 没有吗啡，以阐明阿片类药物对不同组织中潜伏感染的T细胞和小胶质细胞的作用。 2)确定DEG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 使用源自诱导的CD 4 + T细胞和小胶质细胞的分离的CD 4 + T细胞和小胶质细胞的CRISPR活化/干扰实验 多能干细胞，以确定鉴定的DEG与潜伏期建立的功能相关性， 没有吗啡和海洛因3)对来自最后一名患者脾脏的HIV+ CD 4 + T细胞进行蛋白质组学分析 礼品参与者。我们将使用DAb-seq，它允许高覆盖率的基因组测序和同时测序。 分析来自最后礼物参与者的脾脏的CD 4 + T细胞上的细胞表面蛋白表达， 吗啡，以确定前病毒的完整性，其整合位点，和蛋白表达的变化， 阿片类药物使用诱导的潜伏感染细胞。4)同时对RNA、蛋白质和整个HIV基因组进行测序 HIV DNA+细胞。我们将把FIND-Seq开发成一种多模式检测方法， 细胞计数器，并同时对回收的细胞进行基因组学、转录组学和蛋白质组学分析。我们 在艾滋病毒的生物学和高级研究方面， 尖端的微流体、CRISPR和多组学测序技术， 来自“最后礼物队列”和“约翰”的艾滋病毒感染者和阿片类药物使用者临床护理方面的领先专家 霍普金斯HIV临床队列研究。我们期望获得对“潜在分子”的范式转变的洞察力， HIV潜伏期在CNS、淋巴和骨髓中启动、建立和维持的机制 组织和物质使用如何影响这些过程”，并与该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.