Irreversible Proviral Silencing in Myeloid Cells

骨髓细胞中不可逆的前病毒沉默

• 批准号: 10705469
• 负责人: Mario Stevenson
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-02 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705469
• 关键词: Alveolar Macrophages; Anatomy; Attention; Binding; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell Compartmentation; Cell physiology; Cells; Central Nervous System; Chemicals; Clinical; Competence; Development; Dominant-Negative Mutation; Epigenetic Process; Excision; Exhibits; Gene Expression; Genetic Transcription; HIV; HIV-1; HIV/AIDS; I-kappa B Proteins; Individual; Infection; Interruption; Kupffer Cells; Link; Liver; Lung; Macaca; Macrophage; Mediating; Methods; Methylation; Microglia; Modeling; Modification; Myelogenous; Myeloid Cells; NF-kappa B; Natural Killer Cells; Phagocytes; Phylogenetic Analysis; Physiological; Plasma; Population; Process; Production; Proviruses; Refractory; Research; Sampling; T-Lymphocyte; Therapeutic; Viral; Viral reservoir; Viremia; Virion; Virus; Virus Activation; Virus Latency; Virus Replication; antagonist; cancer therapy; cell killing; cytokine; cytotoxic CD8 T cells; epigenetic silencing; experimental analysis; experimental study; heterokaryon; in vivo; induced pluripotent stem cell; inhibitor; molecular clock; monocyte; novel; novel strategies; pharmacologic; resilience; simian human immunodeficiency virus

ABSTRACT: In the pursuit of strategies with which to cure HIV-1 infection, most of the attention has been on mechanisms that govern HIV-1 persistence in CD4+ T cell reservoirs and on approaches to limit persistence in those reservoirs. We recently provided evidence [1] that macrophages enable viral persistence in HIV-1-infected individuals on effective ART. We showed that rebound viremia from individuals undergoing an analytic treatment interruption (ATI) comprised highly macrophage-tropic viruses and we used a novel virion immunocapture method to demonstrate that those macrophage-tropic viruses in post-ATI plasma had a myeloid cell origin. Furthermore, Molecular Clock analysis indicated that macrophage-tropic viruses in plasma pre-dated the treatment interruption as opposed to having been created during the treatment interruption interval. Given these lines of evidence that myeloid cells can serve as viral reservoirs under effective ART, we set about establishing a latency model in primary macrophages. In the pursuit of this model of macrophage latency, we observed that NF-kB inhibition, originally intended to promote synchronous latency, led to irreversible loss of proviral competence- proviruses were refractory to latency reactivating agents (LRAs) even though host cell functions remained intact. On the basis of these observations, we hypothesize that the provirus is epigenetically modified under conditions where NF-kB is limiting and that sustained transcriptional activity is required to counteract epigenetic silencing processes of the host cell. In this proposal, we will assess the mechanisms governing loss of proviral competence. Aim 1: Assess correlates of proviral silencing in NF-kB-depleted macrophages. We will determine the stage in the viral replication cycle at which proviruses are rendered inactive and, through heterokaryon formation, whether dominant negative factors mediate irreversible proviral silencing by NF-kB inhibition. We will examine whether inactive proviruses exhibit evidence of epigenetic modification and methylation and whether inhibiting methylation can maintain proviral responsiveness to reactivation in the face of NF-kB inhibition. Aim 2: Examine whether the proviral silencing created under conditions of NF-kB inhibition can be extended to infected CD4+ T cells from ART-suppressed subjects and to other primary macrophage populations. We will assess whether NF-kB inhibition can irreversibly impede HIV-1 reactivation in CD4+ T cells from individuals on effective ART and whether this proviral silencing can also be achieved in macrophages that are physiologically representative of the major myeloid cell compartments including lung (alveolar macrophages), liver (Kupffer cells), and Central Nervous System (microglia). If the proposed studies are successful, they will provide the rationale for in vivo studies using established SHIV/macaque models of macrophage reservoirs and NF-kB antagonists currently in clinical use.

摘要： 在寻求治愈HIV-1感染的策略时，大部分注意力都集中在 控制HIV-1在CD 4 + T细胞库中持续存在的机制，以及限制HIV-1在CD 4 + T细胞库中持续存在的方法。 这些水库。我们最近提供的证据[1]表明，巨噬细胞使HIV-1感染者体内的病毒持续存在。 我们发现，接受分析性治疗的个体的病毒血症反弹， 中断（ATI）包括高度嗜巨噬细胞的病毒，我们使用了一种新的病毒粒子免疫捕获 方法来证明ATI后血浆中的那些嗜巨噬细胞病毒具有髓样细胞来源。 此外，分子钟分析表明，血浆中的嗜巨噬细胞病毒早于 治疗中断，而不是在治疗中断间隔期间产生。 鉴于这些证据表明，在有效的ART下，骨髓细胞可以作为病毒储存库，我们设定 关于在原代巨噬细胞中建立潜伏期模型。在这个巨噬细胞潜伏期模型的研究中， 我们观察到NF-kB的抑制，最初是为了促进同步潜伏期，导致不可逆的丧失， 前病毒能力-前病毒对潜伏再激活剂（LRA）是难治的，即使宿主细胞 功能保持完整。基于这些观察，我们假设前病毒是表观遗传的， 在NF-kB是限制性的并且需要持续的转录活性的条件下修饰， 抵消宿主细胞的表观遗传沉默过程。在本提案中，我们将评估 管理前病毒能力的丧失。 目的1：评估NF-κ B耗尽的巨噬细胞中前病毒沉默的相关性。我们将确定 病毒复制周期中的一个阶段，在此阶段前病毒失去活性，通过异核体 是否显性负性因子通过NF-κ B抑制介导不可逆的前病毒沉默。我们将 检查无活性前病毒是否表现出表观遗传修饰和甲基化的证据， 抑制甲基化可以维持前病毒在面对NF-κ B抑制时对再活化的反应性。 目的2：检查在NF-kB抑制条件下产生的前病毒沉默是否可以 扩展至ART抑制受试者的受感染CD 4 + T细胞和其他原代巨噬细胞 人口。我们将评估NF-kB抑制是否能不可逆地阻止HIV-1在CD 4 + T细胞中的再活化 以及这种前病毒沉默是否也可以在巨噬细胞中实现， 在生理上代表主要的骨髓细胞区室，包括肺（肺泡巨噬细胞）， 肝脏（枯否细胞）和中枢神经系统（小胶质细胞）。 如果拟议的研究成功，它们将为使用已建立的方法进行体内研究提供依据。 目前临床使用的巨噬细胞储库和NF-kB拮抗剂的SHIV/猕猴模型。