Characterizing HIV-1 reservoirs in the central nervous system

中枢神经系统中 HIV-1 储存库的特征

• 批准号: 10772268
• 负责人: Shibani Sharon Mukerji
• 金额: $ 83.47万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10772268
• 关键词: ATAC-seq; Address; Affect; Anatomy; Anti-Retroviral Agents; Astrocytes; Autopsy; Behavior; Biological Assay; Blood; Blood Vessels; Brain; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cells; Cellular Assay; Central Nervous System; ChIP-seq; Characteristics; Chromatin; Clinical; Clonality; Colon; Combined Modality Therapy; Communicable Diseases; Competence; Cytosine; Epigenetic Process; Evaluation; Frequencies; Functional disorder; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genomics; Goals; HIV; HIV-1; Heterochromatin; Histones; Human; Human Genome; Human body; Immune; Immunohistochemistry; Immunologics; In Situ Hybridization; Individual; Infection; Interruption; Intervention; Investigation; Length; Location; Lymphoid Tissue; Macrophage; Mediating; Methylation; Microglia; Myelogenous; NCOA6 gene; Neurocognitive; Neuroglia; Neurology; Pathogenesis; Patient Care; Patients; Persons; Phylogenetic Analysis; Positioning Attribute; Predisposition; Proviruses; RNA; Research Personnel; Resolution; Role; Sampling; Signal Transduction; Sorting; T-Lymphocyte Epitopes; Technology; Testing; Tissues; Transcription Initiation Site; Untranslated RNA; Viral; Viral reservoir; Virus Diseases; Virus Integration; Work; body system; brain cell; cell type; chromosomal location; comparative; fitness; genome analysis; genome-wide; glial activation; histone modification; ileum; insight; integration site; interdisciplinary collaboration; lymph nodes; neuropathology; neutralizing antibody; next generation sequencing; novel; response; single cell technology; single molecule; viral rebound; virology

Abstract A considerable number of clinical, neuropathological, immunohistochemical and immunological studies suggest that human brain cells, in particular those of myeloid origin, are susceptible to HIV-1 infection; infection of these cells may contribute to neurocognitive dysfunction and viral long-term persistence despite ART. However, among all anatomical tissue locations in the human body, the central nervous system (CNS) arguably represents the most difficult one to access and to evaluate for viral infection and persistence. Recently, we and others have made significant progress in defining viral reservoirs at a single-cell and single-molecule resolution, using a platform of novel next-generation sequencing assays allowing to simultaneously analyzing near full-length proviral sequences, the corresponding chromosomal integration sites and the respective HIV-1 RNA expression profile from individual viral reservoir cells. Using such technologies, we observed evidence for immune-mediated selection mechanisms that enable long-term persistence of viral reservoir cells with features of deep latency, while reservoir cells with higher susceptibility to reactivation signals seemed to be actively selected against; in rare cases of individuals with “elite control”, such selection mechanism resulted in a highly-restricted viral reservoir configuration consisting of intact proviruses located in heterochromatin positions not permissive to viral transcription. Here we will propose to use this established and fully-operational technology pipeline for a detailed analysis of the frequency, clonality and replication competence of proviruses isolated from sorted CNS cells, in particular from myeloid parenchymal microglia, from myeloid perivascular macrophages and astrocytes (Specific Aim 1). In addition, we will utilize novel next-generation sequencing assays for characterizing the chromosomal locations of intact and defective proviruses, and their associated epigenetic chromatin microenvironment, using ATAC-Seq, ChIP-Seq and Methylation-Seq assays (Specific Aim 2). For a functional evaluation of proviruses residing in the CNS, we will subsequently conduct reactivation assays with single patient-derived virally-infected cells from the CNS, allowing us to determine how chromosomal positioning and epigenetic features affect the transcriptional activity of proviruses and their susceptibility to latency-reversing agents (Specific Aim 3). Together, these investigations will provide high-resolution insight into the dynamics of HIV-1 persistence in myeloid CNS cell subsets and may be highly informative for targeted HIV-1 cure interventions.

抽象的 大量临床、神经病理学、免疫组织化学和免疫学研究表明 人类脑细胞，特别是骨髓来源的脑细胞，容易受到 HIV-1 感染； 尽管接受了抗逆转录病毒治疗，细胞仍可能导致神经认知功能障碍和病毒长期持续存在。 在人体的所有解剖组织位置中，中枢神经系统 (CNS) 可以说代表了 最近，我们和其他人已经发现了最难访问和评估病毒感染和持续性的方法。 使用单细胞和单分子分辨率在定义病毒库方面取得了重大进展 新型下一代检测测序平台允许同时分析近全长 原病毒序列、相应的染色体整合位点和各自的 HIV-1 RNA 表达 使用此类技术，我们观察到了免疫介导的证据。 选择机制使具有深潜伏期特征的病毒库细胞能够长期持续存在， 而对重新激活信号更敏感的储存细胞似乎被积极选择； 极少数具有“精英控制”的个体，这种选择机制导致了高度限制的病毒 由位于不允许病毒存在的异染色质位置的完整原病毒组成的储存库构型 在这里，我们将建议使用这个已建立且完全运行的技术管道来进行详细的转录。 分析从分选的 CNS 细胞中分离出的原病毒的频率、克隆性和复制能力， 特别是来自骨髓实质小胶质细胞、来自骨髓血管周围巨噬细胞和星形胶质细胞 （具体目标 1）此外，我们将利用新型下一代测序分析来表征 完整和缺陷原病毒的染色体位置及其相关的表观遗传染色质 微环境，使用 ATAC-Seq、ChIP-Seq 和甲基化-Seq 检测（具体目标 2）。 评估驻留在中枢神经系统中的原病毒，我们随后将使用单一的病毒进行再激活测定 来自中枢神经系统的患者来源的病毒感染细胞，使我们能够确定染色体定位和 表观遗传特征影响原病毒的转录活性及其对潜伏期逆转的敏感性 这些调查将共同提供对动态的高分辨率洞察。 HIV-1 在骨髓 CNS 细胞亚群中持续存在，可能为 HIV-1 靶向治疗提供大量信息 干预措施。