Irreversible Proviral Silencing in Myeloid Cells

摘要： 在寻求治愈HIV-1感染的策略方面，人们的大部分注意力都集中在 控制HIV-1在CD4+T细胞库中持续的机制和限制HIV-1持续存在的方法 那些水库。我们最近提供了证据[1]，表明巨噬细胞能够使HIV-1感染患者的病毒持续存在 个人对有效艺术的关注。我们发现，在接受分析治疗的个体中，病毒血症会反弹。 中断(ATI)由高度嗜巨噬细胞的病毒组成，我们使用了一种新的病毒粒子免疫捕获 方法证实ATI后血浆中的嗜巨噬细胞病毒起源于髓系细胞。 此外，分子时钟分析表明，血浆中的嗜巨噬细胞病毒早于 处理中断，而不是在处理中断间隔期间创建的。 鉴于这些证据表明，在有效的抗逆转录病毒疗法下，髓系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。在追求这种巨噬细胞潜伏期模型的过程中， 我们观察到，抑制核因子-kB最初旨在促进同步潜伏期，导致不可逆转的损失 前病毒能力-前病毒对潜伏期再活化剂(LRA)不敏感，即使宿主细胞 功能完好无损。在这些观察的基础上，我们假设前病毒是表观遗传的 在核因子-kB受到限制且需要持续转录活性的情况下进行修饰 中和宿主细胞的表观遗传沉默过程。在这份提案中，我们将评估这些机制 治理前身能力的丧失。 目的1：评估核因子-kB耗竭的巨噬细胞中前病毒沉默的相关性。我们将决定 病毒复制周期中的一个阶段，在这一阶段前病毒变得不活跃，并通过异核体 形成，显性负面因素是否通过抑制核因子-kB而介导不可逆前病毒沉默。我们会 检查非活动性前病毒是否表现出表观遗传修饰和甲基化的证据，以及是否 抑制甲基化可以在面临核因子-kB抑制的情况下维持对重新激活的前病毒反应性。 目的2：检测在核因子-kB抑制条件下产生的前驱沉默是否可以 延伸到ART抑制受试者感染的CD4+T细胞和其他原代巨噬细胞 人口。我们将评估抑制核因子-kB是否可以不可逆转地阻碍CD4+T细胞中HIV-1的重新激活 来自有效抗逆转录病毒治疗的个体，以及这种前病毒沉默是否也可以在 是包括肺(肺泡巨噬细胞)在内的主要髓系细胞的生理学代表， 肝脏(库普弗细胞)和中枢神经系统(小胶质细胞)。 如果拟议的研究成功，它们将为体内研究提供理论基础，使用已建立的 目前临床使用的巨噬细胞储存库和核因子-kB拮抗剂的SHV/猕猴模型。

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.