Characterizing HIV-1 reservoirs in the central nervous system

摘要 大量的临床、神经病理学、免疫组化和免疫学研究表明， 人类的脑细胞，特别是骨髓来源的脑细胞，易受HIV-1感染; 细胞可能导致神经认知功能障碍和病毒长期持续存在，尽管艺术。 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 最难接近和评估病毒感染和持久性的一种。最近，我们和其他人 在单细胞和单分子分辨率下定义病毒储库方面取得了重大进展， 新一代测序分析平台，允许同时分析近全长 前病毒序列、相应的染色体整合位点和各自的HIV-1 RNA表达 来自单个病毒储库细胞的特征。使用这些技术，我们观察到免疫介导的 选择机制使具有深潜伏特征的病毒储存细胞能够长期持续， 而对再激活信号具有更高敏感性的储库细胞似乎被主动选择; 罕见的情况下，个体与“精英控制”，这种选择机制导致了高度限制的病毒 由位于异染色质位置的完整前病毒组成的储存库构型，不允许病毒感染 转录。在这里，我们将建议使用这一已建立和全面运作的技术管道， 分析从分选的CNS细胞中分离的前病毒的频率、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，我们将利用新的下一代测序分析来表征 完整和缺陷前病毒的染色体定位及其相关的表观遗传染色质 使用ATAC-Seq、ChIP-Seq和甲基化-Seq测定（特异性目的2），在微环境中测定细胞的DNA。某功能 为了评估CNS中存在的前病毒，我们随后将用单个 患者来源的病毒感染的细胞从中枢神经系统，使我们能够确定如何染色体定位和 表观遗传特征影响前病毒的转录活性及其对潜伏期逆转的敏感性 具体目标（3）。总之，这些调查将提供高分辨率的动态洞察力， HIV-1在髓样CNS细胞亚群中的持续存在，可能为靶向HIV-1治疗提供高度信息 干预措施。

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.