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： 潜伏感染的 CD4+ T 细胞被认为是根除 HIV 或功能性治愈的主要障碍， 这些细胞的病毒再激活可能会导致器官炎症和损伤 抗逆转录病毒疗法是开发更有效的艾滋病毒疗法的主要障碍，包括 那些旨在治愈的问题是缺乏对体内潜伏艾滋病毒感染的控制机制的了解。 开发了一种新的“转录分析”方法，可以同时测量转录的程度 通过将这种方法应用于 ART 抑制的细胞，不同的机制可抑制体内 HIV 转录。 在患者中，我们发现一系列对 HIV 转录延伸、远端转录/多聚腺苷酸化的阻断 （完成）和多重剪接是可逆性抑制病毒产生的主要机制 相比之下，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。 一个尚未解答的关键问题是，在小分子中是否存在相同或不同的机制。 具有传染性原病毒的细胞子集，构成真正的潜在病毒库。在本提案的目标 1-3 中， 我们将确定不同机制可逆地阻止个体患者病毒产生的程度 通过使用一种结合了单基因组“全基因组”的新方法，对具有完整或有缺陷的原病毒的细胞 在目标 1 中，我们将应用这些方法来进行“原病毒”测序和单基因组 HIV 转录分析。 来自 ART 抑制患者的血液 CD4+ T 细胞，以确定不同机制的程度 具有完整或有缺陷的原病毒，组成型抑制血液 CD4+ T 细胞中的 HIV 转录，其程度 它们通过离体短期 T 细胞激活而逆转，以及它们在激活方面的差异程度 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 CD4+ T 细胞，以确定组织或 PD-1 是否存在差异 原病毒序列的表达以及个体中可逆抑制病毒产生的机制 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 HIV 的组成型转录和再激活 具有完整和有缺陷的原病毒的细胞中的潜力这三个目标将有助于明确回答这个问题。 什么机制控制艾滋病毒在血液和组织中的体内潜伏期，这是艾滋病毒的一个主要目标 过去 20 年的研究，应该有助于为旨在根除、功能性治愈或 减少治疗艾滋病毒感染的后遗症。