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

确定血液、肠道和淋巴结中 HIV 潜伏感染机制的新单基因组方法

• 批准号: 10611415
• 负责人: Mathias Lichterfeld
• 金额: $ 81.03万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10611415
• 关键词: ATAC-seq; Affect; Agreement; Biological Assay; Blood; Blood Cells; CD4 Positive T Lymphocytes; Cell division; Cells; Chromatin; Coculture Techniques; Data; Detection; Development; Distal; Genetic Transcription; Genome; Goals; HIV; HIV Genome; HIV Infections; HIV therapy; Human; Individual; Infection; Inflammation; Knowledge; Length; Lymphoid Tissue; Measures; Methods; Modeling; Organ; Patients; Poly A; Polyadenylation; Production; Proviruses; RNA Splicing; Rectum; Research; Sampling; Series; T-Cell Activation; Tissues; Transcription Elongation; Transcription Initiation; Viral; Virus; Virus Latency; antiretroviral therapy; effective therapy; ileum; in vivo; individual patient; integration site; latent infection; lymph nodes; novel; novel strategies; novel therapeutics; programmed cell death protein 1; rectal; success; whole genome

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.

项目摘要/摘要： 潜在感染的CD4+ T细胞被认为是消除HIV或功能治愈的主要障碍， 这些细胞的病毒重新激活可能有助于器官注射和损害 抗逆转录病毒疗法。对艾滋病毒更有效疗法的开发的主要障碍，包括 那些旨在治愈的人是缺乏对控制体内潜在艾滋病毒感染的机制的知识。我们 已经开发了一种新的“转录分析”方法，可以轻松衡量 不同的机制抑制了体内HIV转录。通过将这种方法应用于被抑制的细胞 患者，我们发现一系列针对HIV转录伸长的块，远端转录/聚腺苷酸化 （完成），多个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感染细胞表现出强烈而自相矛盾的hiv的障碍 转录。一个关键的未解决的问题是，小型机制在小机制中起作用 具有感染性病毒的细胞子集，构成真正的潜在储层。在本提案的目标1-3中， 我们将确定不同机制可逆地阻止患者的病毒产生的程度 通过使用一种结合两种基因组的新方法，具有完整或有缺陷的病毒的细胞” 原病毒的测序和单基因组HIV转录分析。在AIM 1中，我们将将这些方法应用于 来自艺术抑制的患者的血液CD4+ T细胞，以确定不同机制的程度 组成性地抑制完整或有缺陷的病毒的血液CD4+ T细胞中的HIV转录，该程度 它们被短期T细胞激活的逆转，以及它们在激活中不同的程度 会产生和不产生上清液病毒的细胞。在AIM 2中，我们将将这些研究扩展到PD-1+和PD-1-- 来自血液，淋巴结和肠道的CD4+ T细胞，以确定组织或PD-1是否存在差异 病毒序列的表达和可逆抑制个体病毒产生的机制 具有缺陷且完整的原病毒的细胞。 AIM 3将通过使用一种新方法进一步扩展这些发现 将单个基因组“整个病毒”和整合位点测序与ATAC-SEQ结合在一起 确定整合位点和染色质可及性如何影响HIV转录和重新激活 具有完整且有缺陷的病毒的细胞中的潜力。这三个目标将有助于明确回答 哪些机制控制了血液和组织体内的艾滋病毒潜伏期，这一直是艾滋病毒的主要目标 过去20年的研究，应有助于告知旨在消除，功能治愈或 减少治疗的HIV感染的后遗症。