HIV Infection and Latency In Astrocytes

摘要 联合抗逆转录病毒疗法（cART）大大延长了艾滋病毒感染者的寿命， 改变了艾滋病的面貌。然而，潜伏在细胞/组织中的艾滋病毒阻止了艾滋病病毒的传播。 功能性治愈或完全根除。有限的获得cART和艾滋病毒建立潜伏感染的能力， 使中枢神经系统（CNS）成为独特的HIV储存库。小胶质细胞/巨噬细胞是HIV的主要靶细胞 中枢神经系统的感染，所有这些都可以是生产性的，潜伏性的或持续性的感染。相反，星形胶质细胞被感染， 我们对这些细胞作为HIV潜伏库的理解，以及它们在HIV潜伏期中的作用， 艾滋病毒/神经艾滋病是相当有限的。我们最近发现，细胞与细胞的接触导致成功的艾滋病毒感染， 星形胶质细胞和建立HIV潜伏期在这些细胞中具有极低水平的正在进行的HIV复制。在 此外，我们发现HIV早期基因达特在星形胶质细胞中的表达诱导miR-132并下调甲基化。 CpG结合蛋白2（MeCP 2）是一种染色质重塑表观遗传因子，通过miRNA介导神经毒性。 含有外来体此外，我们已经证明，可卡因激活HIV潜伏感染的星形胶质细胞中的HIV复制 而达特表达与星形胶质细胞中HIV潜伏期的建立有关。最后我们 已经获得了初步数据，即HIV感染者的脑脊液（CSF）中miR-132的表达升高， 轻微认知和运动障碍的受试者。作为我们已发表和初步研究的逻辑延伸，我们 建议描述星形胶质细胞中的HIV感染和潜伏期及其对星形胶质细胞功能的贡献， cART时代的HIV/neuroAIDS。这一建议的基本假设是，艾滋病毒感染的星形胶质细胞构成 CNS中潜伏的HIV储库，并直接导致HIV/神经AIDS。为了验证这一假设，我们建议 解决以下四个相互关联的具体目标：（1）表征细胞-细胞接触介导的HIV感染， （2）阐明HIV在星形胶质细胞中潜伏的分子机制;（3）确定潜伏的HIV对星形胶质细胞的影响。 感染对星形胶质细胞和神经元的影响;和（4）鉴定脑中HIV潜伏期的CSF生物标志物。我们将使用一个 包括使用原代小鼠皮质星形胶质细胞的组合分子、细胞、生物化学和遗传方法 培养物和神经元培养物，多西环素诱导的脑特异性HIV达特转基因小鼠（i达特），原代人胎 脑/星形胶质细胞培养物、脑组织和HIV-1队列的CSF样本。我们预计， 将使我们能够确定星形胶质细胞中HIV潜伏期的调节机制以及这些细胞作为 在cART和可卡因滥用的背景下，艾滋病毒储库。调查结果可能会告知控制和 中枢神经系统HIV宿主的根除策略。关于艾滋病毒感染的大量信息 和发病机制在中枢神经系统/星形胶质细胞和艾滋病毒潜伏期在外周和从我们的初步研究中获得的结果， 研究使这些目标的实现变得切实可行。

ABSTRACT Combination antiretroviral therapy (cART) has considerably prolonged the lifespan of HIV-infected individuals and changed the landscape of the HIV/AIDS disease. However, latent HIV in the cells/tissues has prevented from achieving a functional cure or complete eradication. Limited access to cART and the ability of HIV to establish latent infection have made the central nervous system (CNS) unique HIV reservoirs. Microglia/macrophages are the main target cells for HIV infection in the CNS and all can be productively, latently or persistently infected. In contrast, astrocytes are infected but in a restricted manner; our understanding of these cells as HIV latent reservoirs and their roles of HIV latency in HIV/neuroAIDS is quite limited. We have recently found that cell-cell contact leads to successful HIV infection of astrocytes and establishment of HIV latency in these cells with an extremely low level of ongoing HIV replication. In addition, we have found that expression of HIV early gene Tat in astrocytes induces miR-132 and down-modulates methyl CpG-binding protein 2 (MeCP2), a chromatin-remodeling epigenetic factor and causes neurotoxicity through miRNA- containing exosomes. Moreover, we have shown that cocaine activates HIV replication in HIV latently infected astrocytes through miR-132 expression and that Tat expression is linked to establishment of HIV latency in astrocytes. Lastly, we have obtained the preliminary data that miR-132 expression in the cerebrospinal fluid (CSF) is elevated in HIV-infected subjects with minor cognitive and motor disorder. As a logical extension of our published and preliminary studies, we propose to characterize HIV infection and latency in astrocytes and their contribution to astrocyte function and HIV/neuroAIDS in the era of cART. The underlying hypothesis of this proposal is that HIV-infected astrocytes constitute latent HIV reservoirs in the CNS and directly contribute to HIV/neuroAIDS. To test this hypothesis, we propose to address the following four interrelated specific aims: (1) To characterize cell-cell contact-mediated HIV infection of astrocytes; (2) To elucidate molecular mechanisms of HIV latency in astrocytes; (3) To determine effects of latent HIV infection on astrocytes and neurons; and (4) To identify CSF biomarkers for HIV latency in the brain. We will use a combined molecular, cellular, biochemical, and genetic approach including use of primary mouse cortical astrocyte cultures and neuron cultures, doxycycline-inducible brain-specific HIV Tat transgenic mice (iTat), primary human fetal brain/astrocyte cultures, brain tissues and CSF samples of HIV-1 cohorts in our studies. We anticipate that this proposal will allow us to determine the regulatory mechanisms of HIV latency in astrocytes and the significance of these cells as HIV reservoirs in the ear of cART and in the context of cocaine abuse. The findings will likely inform control and eradication strategies for the HIV reservoirs in the CNS. The enormous amount of information available on HIV infection and pathogenesis in the CNS/astrocytes and HIV latency in the periphery and the results obtained from our preliminary studies make accomplishment of these aims practical.