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

描述(申请人提供)：HIV-1病毒不能仅靠抗逆转录病毒疗法治愈。治愈HIV-1的主要障碍是病毒在细胞内以潜伏但可激活的形式持续存在的能力。HIV-1最典型的细胞储存库是周围器官系统的静止记忆CD4T细胞隔间。然而，在感染期间，大脑很早就被HIV-1定植，大脑中的常驻细胞类型，包括星形胶质细胞和巨噬细胞系细胞(血管周围巨噬细胞和小胶质细胞)经常被感染，并可能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的治疗策略至关重要。我们的广泛假设是，潜伏在星形胶质细胞和脑中非生产性感染的巨噬细胞亚群中的HIV-1长末端重复序列(LTRs)不同于在同一受试者的生产性感染的脑巨噬细胞和外周血单核细胞(PBMC)中检测到的那些序列，并且由与Sp1转录因子结合基序相关的变化定义。此外，这些潜伏的LTRs中Sp1结合的变化导致染色质复合体中的重排，并降低和/或改变LTR被HIV-1Tat蛋白和其他转录激活剂激活的能力。这些假说将在一种新的和临床相关的环境中进行验证，方法是使用从CART治疗患者的脑组织和匹配的PBMC提纯的细胞群体中提取的LTRs进行体外测试。首先，我们将利用尖端的激光捕获显微切割技术和高灵敏的聚合酶链式反应和克隆方案，从纯化的星形胶质细胞和巨噬细胞中获得大量的LTR序列，这些细胞要么是生产性感染的，要么是非生产性感染的。也将从匹配的患者PBMC中生成LTRS以供比较。这些LTrs将被测序，并根据其转录活性进行功能鉴定。根据我们的初步数据，我们预计 星形胶质细胞和非生产性感染的巨噬细胞的LTrs转录活性相对较低，其特征是Sp-1结合基序的序列变化。其次，我们将使用凝胶迁移率改变分析来确定Sp-1蛋白的结合，并通过染色质免疫沉淀分析来确定相关细胞模型中的染色质重塑因子和转录调节因子，以阐明转录活性改变的机制。最后，我们将测试一组HDAC抑制剂在相关细胞模型中激活来自星形胶质细胞和非生产性感染巨噬细胞的潜在LTrs的能力。这些实验将识别具有细胞特异性或广泛活性的化合物，这些化合物可能会为未来旨在从中枢神经系统潜在储存库中根除HIV-1的战略试验提供信息。

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.