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

中心星形胶质细胞和巨噬细胞储库中的转录 HIV-1 潜伏期

基本信息

批准号：
8656447
负责人：
Melissa Churchill
金额：
$ 14.85万
依托单位：
MACFARLANE BURNET INST FOR RES/PUB HLTH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-26 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8656447
关键词：
Antiviral Therapy Area Astrocytes Binding Biological Assay Brain Cell Lineage Cell model Cells Central Nervous System Infections Chromatin Chromatin Remodeling Factor Clinical Cloning Collaborations Complex DNA DNA Sequence Rearrangement Data Development Discipline Drug usage Electrophoretic Mobility Shift Assay Evolution Flushing Future Gene Mutation Goals HIV HIV-1 Histone Deacetylase Inhibitor Infection Life Long Terminal Repeats Microdissection Microglia Neuraxis Organ Pathogenesis Patients Penetration Pericytes Peripheral Peripheral Blood Mononuclear Cell Pharmaceutical Preparations Phenotype Population Protein Binding Protocols documentation Proviruses Public Health Publishing Refractory Research Rest Sp1 Transcription Factor Technology Terminal Repeat Sequences Testing Transcription Coactivator Transcriptional Activation Transcriptional Regulation Variant Viral Virus antiretroviral therapy base body system brain cell brain tissue cell type chromatin immunoprecipitation clinically relevant in vivo macrophage memory CD4 T lymphocyte novel public health relevance purge research study tat Protein

项目摘要

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.

描述（由申请人提供）：HIV-1不能单独通过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库中以潜伏但可激活的形式持续存在的能力。HIV-1最具特征的细胞库是外周器官系统的静息记忆CD 4 + T细胞区室。然而，大脑在感染过程中很早就被HIV-1定殖，并且大脑内的常驻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，并且可以携带潜伏形式的病毒。因此，了解HIV-1在脑星形胶质细胞和巨噬细胞储库中潜伏的机制对于HIV-1治疗策略至关重要。我们广泛的假设是，潜伏的HIV-1长末端重复序列（LTRs）持续在星形胶质细胞和非生产性感染的巨噬细胞在大脑中的子集是不同的生产性感染的脑巨噬细胞和外周血单核细胞（PBMC）的相同的主题，并定义与Sp1转录因子结合基序相关的改变。此外，这些潜伏LTR中的Sp1结合改变导致染色质复合物中的重排和LTR被HIV-1达特蛋白和其他转录激活因子激活的能力降低和/或改变。这些假设将在一种新的临床相关环境中通过离体试验进行检验，该离体试验使用来自cART治疗患者脑组织和匹配PBMC纯化细胞群的LTR。首先，我们将利用尖端的激光捕获显微切割技术和高灵敏度的PCR和克隆协议，以产生一个大银行的LTR序列从纯化群体的星形胶质细胞，巨噬细胞，无论是生产性感染或非生产性感染。还将从匹配的患者PBMC中生成LTR以进行比较。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，我们预计来自星形胶质细胞和非生产性感染的巨噬细胞的LTR将具有相对降低的转录活性，其特征在于Sp-1结合基序的序列改变。其次，我们将阐明改变转录活性的机制，使用电泳迁移率变动分析，以表征Sp-1蛋白结合，并通过染色质免疫沉淀分析，以确定贡献染色质重塑因子和转录调节因子，使用相关的细胞模型。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伏LTR的能力。这些实验将确定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的战略试验提供信息，这些试验旨在从中枢神经系统的潜伏储库中根除HIV-1。