Innate sensing of HIV in microglia

项目摘要 尽管有抑制性联合抗逆转录病毒治疗（cART），HIV感染仍然存在于组织库中。为 例如，在中枢神经系统（CNS）中，已经在cART中观察到病毒转录物的表达。 抑制患者，这可能有助于慢性免疫激活，这是艾滋病毒相关免疫缺陷的主要驱动因素。 神经认知障碍（HAND）。持续的炎症被认为会导致免疫功能障碍， 免疫控制机制的退化，可能导致病毒持续存在。因此，似乎有一个 炎症和病毒持续存在的正反馈回路。为了减少病毒储存库的大小， 因此，了解导致慢性炎症的机制至关重要。 小胶质细胞，CNS驻留的微神经胶质细胞，被认为是HIV-1的主要细胞库，因为它们的 自我更新能力。我们最近证明，含HIV内含子RNA的细胞质表达 在iPSC（诱导多能干细胞）衍生的细胞中，ICRNA诱导的IFN-I依赖性促炎反应 人类小胶质细胞。因此，我们假设在HIV感染的小胶质细胞中， 导致持续性炎症和免疫功能障碍，导致免疫控制能力下降 机制，并有助于病毒的持久性。然而，迄今为止，艾滋病毒感染如何触发小胶质细胞 激活以及小胶质细胞激活是否影响病毒持久性仍不清楚。在这份提案中，我们将调查 HIV-icRNA诱导小胶质细胞先天免疫激活的机制及其对 免疫功能障碍，具有以下目的。在Aim 1中，我们将鉴定感受HIV icRNA的宿主机制 在小胶质细胞中使用两种互补的质谱（MS）为基础的方法与博士合作。 威斯康星大学麦迪逊分校的Sherer。我们将通过杂交来纯化HIV-icRNA相关蛋白 洁净.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。然后我们将进行MS分析以识别传感器。我们将确认 这些候选蛋白在使用人iPSC衍生的小胶质细胞的小胶质细胞活化中的作用。在AIM 2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iPSC-小胶质细胞中的HIV感染导致可溶性因子如促炎细胞因子的产生， 补体4A和肽基精氨酸脱亚胺酶2。然后，我们将通过CRISPR/Cas9敲除候选基因， 在iPSC中，然后分化为小胶质细胞，并测试KO小胶质细胞中的HIV感染是否导致功能障碍。 我们希望这项提案的成功完成不仅有助于加深我们对HIV RNA宿主的认识， 相互作用，而且有助于未来开发新的治疗方式，减少HIV RNA- 诱导免疫激活和病毒在CNS中的持续存在。

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.