Innate sensing of HIV in microglia

项目总结 尽管进行了抑制性联合抗逆转录病毒疗法(CART)，但艾滋病毒感染仍然存在于组织储备库中。为 例如，在中枢神经系统(CNS)中，已观察到病毒转录本在CART中的表达。 受抑制的患者，这可能导致慢性免疫激活，这是艾滋病毒相关的主要驱动因素 神经认知障碍(手)。持续的炎症被认为会导致免疫功能障碍和 免疫控制机制的退化，可能导致病毒的持久性。因此，似乎有一种 炎症和病毒持久性的正反馈循环。为了减少病毒库的大小并实现 因此，了解导致慢性炎症的机制是至关重要的。 居住在中枢神经系统的小胶质细胞被认为是HIV-1的主要细胞储存库，因为它们 自我更新能力。我们最近证实了含有HIV内含子的RNA的细胞质表达 ICRNAs诱导IPSC来源的干扰素-I依赖的促炎反应 人类的小胶质细胞。因此，我们假设HIV感染的小胶质细胞中有新的HIV icRNA表达 导致持续的炎症和免疫功能障碍，导致免疫控制能力下降 机制和对病毒持久性的贡献。然而，迄今为止，艾滋病毒感染是如何触发小胶质细胞的 激活以及小胶质细胞的激活是否影响病毒的持久性仍不清楚。在这份提案中，我们将调查 HIV-icRNA诱导小胶质细胞天然免疫激活的机制及其在脑损伤中的作用 免疫功能障碍的目标如下。在Aim1中，我们将识别感知HIV icRNA的宿主机制 与Dr合作，使用两种基于互补质谱学(MS)的方法对小胶质细胞进行研究。 威斯康星大学麦迪逊分校的谢勒说。我们将通过杂交的方式纯化HIV-icRNA相关蛋白 净化。此外，我们将使用生物素连接酶介导的邻近标记策略来鉴定宿主蛋白 与HIV-1 icRNA密切接触。然后，我们将执行MS分析以识别传感器。我们将验证该角色 使用人iPSC来源的小胶质细胞激活这些候选蛋白。在AIM2中，我们将 研究HIV感染的小胶质细胞产生的可溶性因子在免疫功能障碍中的作用。我们将测试 如果HIV感染IPSC-小胶质细胞导致促炎细胞因子等可溶性因子的产生， 补体4A和肽基精氨酸脱亚氨酶2。然后用CRISPR/Cas9敲除候选基因 在IPSCs中，然后分化为小胶质细胞，并测试HIV感染是否会导致KO小胶质细胞功能障碍。 我们希望这项提案的成功完成不仅将有助于加深我们对艾滋病毒RNA宿主的了解 相互作用，但也有助于未来开发新的治疗方式，减少艾滋病毒RNA- 诱导中枢神经系统内的免疫激活和病毒持续存在。

PROJECT SUMMARY Despite suppressive combination anti-retroviral therapy (cART), HIV infection persists in tissue reservoirs. For example, in the central nervous system (CNS), expression of viral transcripts has been observed in cART- suppressed patients, which likely contributes to chronic immune activation, the chief driver of HIV-associated neurocognitive disorders (HAND). Persistent inflammation is presumed to cause immune dysfunction and degradation of immune control mechanisms, likely contributing to virus persistence. Thus, there seems to be a positive feedback loop of inflammation and viral persistence. To reduce the size of viral reservoir and achieve a functional HIV cure, therefore, it is crucial to understand mechanisms that contributes to chronic inflammation. Microglia, the CNS-resident microphages, are thought to be a major cellular reservoir of HIV-1 because of their self-renewal capacity. We recently demonstrated that cytoplasmic expression of HIV intron-containing RNAs (icRNAs) induced IFN-I-dependent pro-inflammatory responses in iPSC (induced pluripotent stem cell)-derived human microglia. We therefore hypothesize that de novo HIV icRNA expression in HIV-infected microglia drives persistent inflammation and immune dysfunction, leading to degradation of immune control mechanisms and contributing to virus persistence. To date, however, how HIV infection triggers microglia activation and if microglia activation affects viral persistence remain unclear. In this proposal, we will investigate mechanisms of HIV-icRNA-induced innate immune activation in microglia and their contribution to immune dysfunction with the following aims. In Aim1, we will identify the host machinery sensing HIV icRNA in microglia using two complementary mass spectrometry (MS)-based approaches in collaboration with Dr. Sherer at University of Wisconsin-Madison. We will purify HIV-icRNA-associated proteins by hybridization purification. In addition, we will use biotin ligase-mediated proximity labeling strategy to identify host proteins in close contact with HIV-1 icRNA. We will then perform MS analysis to identify the sensor. We will validate the role of these candidate proteins in microglia activation using human iPSC-derived microglia. In Aim2, we will investigate the role of soluble factors produced from HIV-infected microglia in immune dysfunction. We will test if HIV infection in iPSC-microglia leads to production of soluble factors such as pro-inflammatory cytokines, complement 4A, and peptidyl arginine deiminase 2. We will then knock out candidate genes by CRISPR/Cas9 in iPSCs, followed by differentiation to microglia, and test if HIV infection in KO microglia leads to dysfunction. We hope that successful completion of this proposal will not only help deepen our knowledge on HIV RNA-host interaction, but also contribute to future development of novel therapeutic modalities that reduce HIV RNA- induced immune activation and virus persistence in the CNS.