Cell-lineage specific epigenomic determinants of HIV latency in humanized mouse brain and blood

项目摘要 人类免疫缺陷病毒1型(HIV-1)感染影响全球3800万人， 由于早期建立了病毒仍然潜伏的水库，因此仍然无法治愈。HIV-1病毒进入 脑部在感染后的前两周内，与随行的神经系统症状一起观察 急性HIV疾病中的中枢神经系统(CNS)生物标志物。中枢神经系统中的70亿个小胶质细胞是 在成人大脑和中枢神经系统中感染艾滋病毒的主要细胞类型，代表一种 大的潜在储油层位置。此外，大脑是与艾滋病毒相关的负担最高的器官之一 疾病。艾滋病毒相关性神经认知障碍(HAND)影响20%-50%的艾滋病毒(PWH)患者， 在联合抗逆转录病毒治疗(CART)时代，占主导地位的是较温和的手部形式。重要的是，完整的艾滋病毒 尽管CART抑制了病毒传播，但前病毒仍在大脑中存在。尽管如此，人们对独一无二的 管理艾滋病毒在大脑中的激活和潜伏的调节机制。根据我们最近的细胞研究，在 人死后脑、炎症相关的小胶质细胞转录本的重新编程和3D 基因组(染色体构象)是与病毒感染和脑细胞整合有关的关键因素 与脑炎相关的晚期感染。然而，非脑炎感染的人脑， 除了显示转录信号表明小胶质细胞与神经元的相互作用中断之外 Synapse，提供了很少的关于表观基因组和其他控制病毒激活和 大脑中的潜伏期。在这里，作为了解控制HIV潜伏期的分子机制的第一步 在人性化的小鼠大脑中，我们将探索一个极其创新的分子工具箱，在 单细胞水平，受感染的小胶质细胞和其他活跃表达HIV的髓系细胞，并将它们从 不表达HIV的受感染细胞(潜伏)。我们将使用此工具箱进行高级实验方法，以 定量测试分子、表观遗传学和药理学干预措施，旨在减少 人源化的HIV+脑、脾和血液。我们将采用一种新的基因方法，称为增强型艾滋病毒- 诱发谱系追踪(E-HILT)揭示中枢神经系统潜伏期建立的频率和动力学 在单细胞分辨率下。我们将在细胞培养和人源化小鼠脑和脾/血中定义 单细胞水平分辨率，有效感染与潜伏感染的比例表示 受遗传或药物诱导的小胶质细胞、淋巴细胞和其他外周髓系细胞 干扰染色质结合的消音器，包括人类沉默中心(HUSH)/CTIP2- KAP1/KMT1E/SETDB1抑制组蛋白甲基转移酶复合体和更广泛的组蛋白H3-赖氨酸9 甲基化(H3K9me)相关的抑制性染色质重塑。这项研究将揭示潜伏期的程度 在体内的原代小胶质细胞中，探索转录组、表观基因组和染色3D结构 支持潜伏期，并探索染色质调节药物对这些细胞状态的影响。

Project Summary Human immunodeficiency virus type 1 (HIV-1) infection affects more than 38 million people worldwide and remains incurable due to the early establishment of reservoirs where the virus remains latent. HIV-1 enters the brain within the first two weeks of infection, and neurologic symptoms have been observed with accompanying central nervous system (CNS) biomarkers in acute HIV disease. The seven billion microglial cells in the CNS are the primary cell type infected by HIV in the adult human brain, and in the central nervous system represents a large potential reservoir site. Additionally, the brain is one of the organs with the highest burden of HIV-associated disease. HIV-associated neurocognitive disorder (HAND) affects 20-50% of people with HIV (PWH), with the milder forms of HAND predominating in the era of combined antiretroviral therapy (cART). Importantly, intact HIV proviruses persist in the brain despite viral suppression with cART. Despite this, little is known about the unique regulatory mechanisms governing HIV activation and latency in the brain. According to our recent cell studies in human postmortem brain, inflammation-associated reprogramming of microglial transcriptomes and 3D genomes (chromosomal conformations) is a key factor linked to viral infection and integration in brain cells during advanced stages of infection associated with encephalitis. However, non-encephalitic infected human brain, other than showing transcriptomic signatures indicative for disrupted interactions of microglia with the neuronal synapse, provides little information about the epigenomic and other determinants governing viral activation and latency in the brain. Here, as a first step towards understanding molecular mechanisms governing HIV latency in the humanized mouse brain, we will explore an extremely innovative molecular toolbox differentiating, on the single cell level, infected microglia and other myeloid cells actively expressing HIV, and separating them from infected cells not expressing HIV (latent). We will use this toolbox for advanced experimental approaches to quantitatively test molecular, epigenetic, and pharmacological interventions aimed at reducing the reservoir of humanized HIV+ brain, spleen, and blood. We will employ a novel genetic approach called enhanced HIV- induced lineage tracing (E-HILT) to reveal the frequency and kinetics of the establishment of latency in the CNS at the single cell resolution. We will define, in cell culture, and in humanized mouse brain and spleen/blood at single cell level resolution, the proportional representation of productively infected versus latently infected microglia and lymphocytes and other peripheral myeloid cells subject to genetically or pharmacologically induced disruption of chromatin-bound silencers, including the Human Silencing Hub (HUSH)/CTIP2- KAP1/KMT1E/SETDB1 repressive histone methyltransferase complex and more, broadly, histone H3-lysine 9 methylation (H3K9me)-associated repressive chromatin remodeling. The study will uncover the degree of latency in primary microglial cells in vivo, explore the transcriptome, epigenomic and chromatic 3D architecture that supports latency and explore the effects of chromatin modulating drugs on these cell states.