Transcriptional HIV-1 latency in astrocyte and macrophage reservoirs of the centr

描述（由申请人提供）：HIV-1不能单靠抗逆转录病毒治疗治愈。治愈HIV-1的主要障碍是该病毒在细胞储存库中以潜伏但可激活的形式持续存在的能力。最具特征的HIV-1细胞储存库是外周器官系统的静息记忆CD4+ t细胞室。然而，在感染过程中，大脑很早就被HIV-1定植，并且大脑内的驻留细胞类型包括星形胶质细胞和巨噬细胞谱系细胞（血管周围巨噬细胞和小胶质细胞）经常被感染，并且可以隐藏病毒的潜伏形式。因此，了解大脑星形胶质细胞和巨噬细胞储存库中HIV-1潜伏期的机制对于HIV-1治疗策略至关重要。我们的广泛假设是，潜伏的HIV-1长末端重复序列（LTRs）持续存在于星形胶质细胞和大脑中非生产性感染的巨噬细胞亚群中，与在相同受试者的生产性感染的脑巨噬细胞和外周血单核细胞（PBMC）中检测到的序列不同，并通过Sp1转录因子结合基序的相关改变来定义。此外，这些潜伏LTR的Sp1结合改变导致染色质复合体的重排，降低和/或改变LTR被HIV-1 Tat蛋白和其他转录激活因子激活的能力。这些假设将在一个新的和临床相关的环境中进行检验，通过体外试验，使用从cart治疗患者脑组织纯化的细胞群和匹配的PBMC中获得的ltr。首先，我们将利用尖端的激光捕获显微解剖技术和高灵敏度的PCR和克隆技术，从纯化的星形胶质细胞和巨噬细胞群体中获得大量的LTR序列，这些细胞要么是生产性感染，要么是非生产性感染。LTRs也将从匹配的患者PBMC中生成，用于比较。将对这些ltr进行测序，并对其转录活性进行功能表征。根据我们的初步数据，我们预计

DESCRIPTION (provided by applicant): HIV-1 cannot be cured by antiretroviral therapies alone. The major obstacle to curing HIV-1 is the ability of the virus to persist in a latent but activatable form within cellular reservoirs. The best-characterized cellular reservoir of HIV-1 is the resting memory CD4+ T-cell compartment of peripheral organ systems. However, the brain becomes colonized with HIV-1 very early during infection, and resident cell types within the brain including astrocytes and macrophage-lineage cells (perivascular macrophages and microglia) are frequently infected and can harbor latent forms of the virus. Understanding the mechanisms of HIV-1 latency in astrocyte and macrophage reservoirs of the brain is therefore critical for HIV-1 cure strategies. Our broad hypothesis is that latent HIV-1 long terminal repeat sequences (LTRs) persisting in astrocytes and a non-productively infected subset of macrophages in the brain are distinct to those detected in productively infected brain macrophages and peripheral blood mononuclear cells (PBMC) of the same subjects, and are defined by alterations associated with the Sp1 transcription factor binding motif. Furthermore, Sp1 binding alterations in these latent LTRs result in rearrangements in chromatin complexes and reduced and/or altered ability of the LTR to be activated by the HIV-1 Tat protein and other transcriptional activators. These hypotheses will be tested in a novel and clinically relevant setting by ex vivo assays using LTRs derived from populations of cells purified from brain tissues of cART-treated patients, and from matched PBMC. First, we will utilize cutting edge lase capture microdissection technology and highly sensitive PCR and cloning protocols to generate a large bank of LTR sequences obtained from purified populations of astrocytes, and of macrophages that are either productively infected or non-productively infected. LTRs will also be generated from matched patient PBMC for comparison. These LTRs will be sequenced and characterized functionally for their transcriptional activity. From our preliminary data, we expect that the LTRs from astrocytes and non- productively infected macrophages will have comparatively reduced transcriptional activity characterized by sequence alterations in the Sp-1 binding motif. Second, we will elucidate the mechanisms of altered transcriptional activity using electrophoretic mobility shift assays to characterize Sp-1 protein binding, and by chromatin immunoprecipitation assays to determine the contributing chromatin remodeling factors and transcriptional regulators, using relevant cellular models. Finally, we will test a panel of HDAC inhibitors for their ability to activate latent LTRs derived from both astrocytes and non-productively infected macrophages in relevant cellular models. These experiments will identify the compounds that have either cell specific or broad activity that may inform future strategic trials aimed at eradicating HIV-1 from latent reservoirs of the central nervous system.