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

项目概要/摘要： 潜伏感染的CD 4 + T细胞被认为是HIV根除或功能性治愈的主要障碍， 这些细胞的病毒再活化可能导致器官炎症和损伤， 抗逆转录病毒疗法艾滋病是开发更有效的艾滋病毒疗法的主要障碍， 这些治疗的目的，是缺乏知识的机制，管理潜伏的艾滋病毒感染的体内。我们 已经开发了一种新的“转录谱”方法，可以同时测量 不同的机制抑制体内HIV转录。通过将这种方法应用于ART抑制的细胞， 我们发现一系列阻断HIV转录延伸、远端转录/多聚腺苷酸化的基因， （完成）和多重剪接是可逆地抑制病毒产生的主要机制， 血相反，直肠中的HIV感染细胞显示出对HIV启动的强烈和矛盾的阻断 转录。一个关键的未回答的问题是，在小的国家， 具有感染性前病毒的细胞亚群，其构成真正的潜伏储库。在本提案的目标1-3中， 我们将确定不同机制在个体患者中可逆性阻断病毒产生的程度， 细胞与完整或有缺陷的前病毒通过使用一种新的方法， 前病毒测序和单基因组HIV转录谱分析。在目标1中，我们将这些方法应用于 ART抑制患者的血液CD 4 + T细胞，以确定不同机制 组成性抑制具有完整或缺陷前病毒的血液CD 4 + T细胞中的HIV转录， 它们通过离体短期T细胞活化而逆转，以及它们在活化T细胞中的不同程度。 产生和不产生上清液病毒的细胞。在目标2中，我们将这些研究扩展到PD-1+和PD-1-。 来自血液、淋巴结和肠道的CD 4 + T细胞，以确定组织或PD-1是否存在差异 在前病毒序列中的表达以及在个体中可逆地抑制病毒产生的机制 有缺陷的和完整的前病毒的细胞。Aim 3将通过使用一种新方法进一步扩展这些发现， 将单基因组“全原病毒”和整合位点测序与ATAC-seq相结合， 确定整合位点和染色质可及性如何影响组成型HIV转录和再活化 在具有完整和有缺陷的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： 是什么机制决定了艾滋病毒在血液和组织中的体内潜伏期，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，功能性治愈，或 减少艾滋病毒感染治疗后的后遗症。

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.