Novel single genome approaches to determine the mechanisms of HIV latent infection in blood, gut, and lymph nodes

项目摘要/摘要： 潜伏感染的CD4T细胞被认为是根除艾滋病毒或治愈功能的主要障碍， 从这些细胞中重新激活病毒可能有助于观察到的器官炎症和损伤 抗逆转录病毒治疗。开发更有效的艾滋病毒治疗方法的主要障碍包括 那些旨在治愈的问题是缺乏对体内潜伏的艾滋病毒感染机制的了解。我们 已经开发出一种新的“转录简档”方法，可以同时测量 不同的机制在体内抑制HIV转录。通过将这种方法应用于ART抑制的细胞 患者中，我们发现了一系列阻断HIV转录延伸、远端转录/多聚腺苷的作用 (完成)和多重剪接是可逆地抑制病毒在 血。相反，直肠中感染艾滋病毒的细胞对艾滋病毒的启动表现出强烈而矛盾的阻碍。 抄写。一个关键的悬而未决的问题是，是相同的机制还是不同的机制在小企业中运作 具有感染性前病毒的细胞亚群，它们构成真正的潜伏蓄水池。在本提案的目标1-3中， 我们将确定不同机制在多大程度上可逆地阻止个别患者的病毒产生 使用一种新的方法结合单一基因组的完整或缺陷前病毒 前病毒“测序和单基因组艾滋病毒转录图谱。在目标1中，我们将把这些方法应用于 从ART抑制患者外周血中CD4T细胞确定不同机制的程度 在前病毒完整或缺陷的情况下，结构性地抑制血液CD4T细胞中HIV的转录，程度到 它们在体外被短期T细胞激活逆转，以及它们在激活程度上的不同 产生和不产生上清液病毒的细胞。在目标2中，我们将把这些研究扩展到PD-1和PD-1- 从血液、淋巴结和肠道中提取CD4T细胞，以确定是否存在组织或PD-1的差异 前病毒序列的表达及其在个体中可逆抑制病毒产生的机制 有缺陷和完整的前病毒的细胞。目标3将通过使用一种新的方法进一步扩展这些发现 将单基因组“完整前病毒”和整合位点测序与atac-seq相结合，以 确定整合部位和染色质的可及性如何影响HIV的组成性转录和重新激活 具有完整和缺陷前病毒的细胞中的潜力。这三个目标将有助于明确回答以下问题 是什么机制控制了艾滋病毒在血液和组织中的体内潜伏，这一直是艾滋病毒的主要目标 过去20年的研究，并应有助于为旨在根除、功能治愈或 减少治疗艾滋病毒感染的后遗症。

Project Summary/Abstract: Latently-infected CD4+ T cells are thought to be the main barrier to HIV eradication or functional cure, and viral reactivation from these cells may contribute to the organ inflammation and damage observed on antiretroviral therapy. A major impediment to the development of more effective therapies for HIV, including those aimed at cure, is the lack of knowledge about the mechanisms that govern latent HIV infection in vivo. We have developed a new “transcription profiling” approach that can simultaneously measure the degree to which different mechanisms inhibit HIV transcription in vivo. By applying this approach to cells from ART-suppressed patients, we found that a series of blocks to HIV transcriptional elongation, distal transcription/polyadenylation (completion), and multiple splicing are the main mechanisms that reversibly suppress virus production in the blood. In contrast, HIV-infected cells in the rectum showed a strong and paradoxical block to initiation of HIV transcription. A critical unanswered question is whether the same or different mechanisms operate in the small subset of cells with infectious proviruses, which constitute the true latent reservoir. In aims 1-3 of this proposal, we will determine the degree to which different mechanisms reversibly block virus production in individual patient cells with intact or defective proviruses by using a novel approach that combines both single genome “whole provirus” sequencing and single genome HIV transcription profiling. In aim 1, we will apply these methods to blood CD4+ T cells from ART-suppressed patients to determine the degree to which different mechanisms constitutively inhibit HIV transcription in blood CD4+ T cells with intact or defective proviruses, the degree to which they are reversed by short term T cell activation ex vivo, and the degree to which they differ in activated cells that do and do not produce supernatant virus. In aim 2, we will extend these studies to PD-1+ and PD-1- CD4+ T cells from blood, lymph node, and gut to determine whether there are differences by tissue or PD-1 expression in proviral sequences and the mechanisms that reversibly suppress virus production in individual cells with defective and intact proviruses. Aim 3 will further extend these findings by using a new method that combines single genome “whole provirus” and integration site sequencing, in combination with ATAC-seq, to determine how integration site and chromatin accessibility affect constitutive HIV transcription and reactivation potential in cells with intact and defective proviruses. These 3 aims will help definitively answer the question of what mechanisms govern HIV latency in vivo in the blood and the tissues, which has been a major goal of HIV research for the last 20 years and should help inform new therapies aimed at eradication, functional cure, or reducing the sequelae of treated HIV infection.