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

人源化小鼠大脑和血液中HIV潜伏期的细胞谱系特异性表观基因组决定因素

• 批准号: 10747752
• 负责人: Schahram Akbarian
• 金额: $ 72.93万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-25 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10747752
• 关键词: 3-Dimensional; Acute; Adult; Affect; Aftercare; Architecture; Autopsy; Binding; Biological Markers; Biological Models; Blood; Brain; Cell Culture System; Cell Culture Techniques; Cell Lineage; Cells; Central Nervous System; Chromatin; Chromium; Complex; Disease; Dominant-Negative Mutation; Drug Modulation; DsRed; Encephalitis; Epigenetic Process; Experimental Models; Frequencies; Genetic; Genetic Engineering; Genome; Genome Mappings; Genomics; Goals; HIV; HIV Infections; HIV-1; HIV-associated neurocognitive disorder; Histone H3; Histones; Human; Immune; Immune system; In Vitro; Infection; Inflammation; Intervention; Kinetics; Life Cycle Stages; Link; Long Terminal Repeats; Lymphocyte; Lysine; Methionine; Methylation; Microglia; Molecular; Molecular Conformation; Mus; Myelogenous; Myeloid Cells; Neurologic Symptoms; Neurons; Organ; Pathogenicity; Peripheral; Persons; Population; Productivity; Proviruses; Regimen; Reporter; Repression; Resolution; Retrotransposon; Retroviridae; SETDB1 gene; Shock; Site; Sorting; Spleen; Synapses; System; Techniques; Testing; Transgenes; Viral; Viral reservoir; Virus; Virus Activation; Virus Diseases; Virus Integration; Virus Latency; Work; Xenograft procedure; antiretroviral therapy; brain cell; cell type; chromatin remodeling; derepression; design; epigenomics; genetic approach; histone methyltransferase; humanized mouse; in vivo; innovation; integration site; mouse model; novel; pharmacologic; response; single-cell RNA sequencing; tissue culture; transcriptome; transcriptomic profiling; transcriptomics

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.

项目摘要 人类免疫缺陷病毒1型（HIV-1）感染影响着全世界3 800多万人， 由于病毒在早期就形成了潜伏的宿主，因此仍然无法治愈。HIV-1进入 在感染的头两周内，大脑出现了神经系统症状，并伴有 中枢神经系统（CNS）生物标志物在急性HIV疾病中的作用。中枢神经系统中的70亿个小胶质细胞 在成年人大脑和中枢神经系统中被HIV感染的主要细胞类型代表了一种 大型潜在水库。此外，大脑是艾滋病毒相关疾病负担最高的器官之一。 疾病HIV相关的神经认知障碍（HAND）影响20-50%的HIV感染者（PWH）， 在联合抗逆转录病毒疗法（cART）时代占主导地位的较温和形式的HAND。重要的是，完整的艾滋病毒 尽管用cART抑制病毒，但前病毒仍在脑中持续存在。尽管如此，人们对这种独特的 控制HIV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， 人类死后脑，炎症相关的小胶质细胞转录组重编程和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， 除了显示指示小胶质细胞与神经元的相互作用被破坏的转录组特征之外 突触，提供了很少的信息表观基因组和其他决定因素控制病毒活化， 大脑中的潜伏期。在这里，作为了解HIV潜伏期分子机制的第一步， 在人源化小鼠大脑中，我们将探索一个极其创新的分子工具箱，区分 在单个细胞水平上，感染的小胶质细胞和其他活跃表达HIV的骨髓细胞，并将它们与 感染细胞不表达HIV（潜伏）。我们将使用此工具箱进行高级实验方法， 定量测试分子，表观遗传和药理学干预，旨在减少水库， 人源化HIV+脑、脾和血液。我们将采用一种新的遗传方法，称为增强型艾滋病毒- 诱导谱系追踪（E-HILT），以揭示CNS中潜伏期建立的频率和动力学 单细胞分辨率。我们将在细胞培养中以及在人源化小鼠脑和脾/血液中定义， 单细胞水平分辨率，生产性感染与潜伏性感染的比例代表 小胶质细胞和淋巴细胞以及其他外周髓样细胞受到遗传或免疫诱导， 破坏染色质结合的沉默剂，包括人类沉默中心（HUSH）/CTIP 2- KAP 1/KMT 1 E/SETDB 1抑制性组蛋白甲基转移酶复合物和更广泛的组蛋白H3-赖氨酸9 甲基化（H3 K9 me）相关的抑制性染色质重塑。该研究将揭示延迟的程度 在体内的原代小胶质细胞中，探索转录组、表观基因组和染色质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。