Investigation of Latency Promoting Genes (LPGs) in HIV Oral Reservoir Cells

HIV 口腔储存细胞潜伏期促进基因 (LPG) 的研究

• 批准号: 9283243
• 负责人: Jian Zhu
• 金额: $ 38.04万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9283243
• 关键词: AIDS/HIV problem; Affect; Alpha Cell; Bioinformatics; Biometry; Biostatistical Methods; Breast Feeding; CD4 Positive T Lymphocytes; CRISPR screen; CRISPR/Cas technology; Cell Death; Cell model; Cells; ChIP-seq; Chemicals; Data Set; Disease; Evaluation; Event; Future; Genes; Genetic; Genetic Transcription; Genital system; Genomic approach; Goals; HIV; HIV-1; Highly Active Antiretroviral Therapy; Histone Deacetylase Inhibitor; Individual; Infection; Integration Host Factors; Investigation; Libraries; Mediating; Methods; Molecular; Molecular Chaperones; Nucleosomes; Ontology; Oral; Oral cavity; Pathway Analysis; Peripheral; Pharmacology; Phenotype; Phosphotransferases; Positive Transcriptional Elongation Factor B; Predisposition; Proteins; Proviruses; RNA Interference; RNA interference screen; Reporting; Resources; Rest; Role; Screening Result; Site; Surface; Test Result; Testing; Tonsil; Translating; Validation; Vorinostat; antiretroviral therapy; base; functional genomics; gene discovery; genome wide association study; genome-wide; genomic tools; inhibitor/antagonist; interest; killings; knock-down; novel; oral HIV; public health relevance; purge; reactivation from latency; screening

 DESCRIPTION (provided by applicant): In the era of HAART, HIV-1 persists as integrated, latent proviruses in reservoir cells of infected individuals, as a major obstacle for cure. Tonsillr CD4+ T cells are a major HIV-1 oral reservoir. Understanding host factors regulating HIV-1 latency in these cells and identify cell-specific latency promoting genes (LPGs) will be useful to develop new latency reversing agents (LRAs) for elimination of HIV-1 oral reservoirs. Our earlier RNAi screens successfully identified LPGs that restrict HIV-1 transcription and facilitate its latency, including BRD4 and SUPT16H. In this proposal, we propose to further characterize these two LPGs in HIV-1 latently infected tonsillar CD4+ T cells (AIM 1 and 2). Their contribution to HIV-1 latency, particularly in oral reservoir cells, is largely unknown and worthy f further characterization. Using our established pipeline, we will also be interested to identify ne LPGs (AIM 3). We believe our proposed study is comprehensive and allows a systematic view how LPGs contribute to HIV-1 latency. AIM 1: We are one of the leading groups to identify BRD4 as a LPG, and we will further characterize BRD4 in HIV-1 latently infected tonsillar CD4+ T cells. We will first create a HIV-1 latency cell model using primary tonsillar CD4+ T cells. BRD4 will be depleted to determine how it affects HIV-1 latency/reactivation. Our preliminary results indicated that P-TEFb mediates BRD4's effect, so we will further determine the impact of BRD4 on P-TEFb activity in tonsillar CD4+ T cells. To test the feasibility to target BRD4 pharmacologically for purging latent HIV-1, we will evaluate a set of new promising BET inhibitors to revert HIV-1 latency. AIM 2: SUPT16H is our newly identified LPG, so we will study it in HIV-1 oral reservoir cells as well. We will determine whether depletion of SUPT16H in tonsillar CD4+ T cells may affect HIV-1 latency/reactivation. We will also use ChIP-seq approach to determine the genome-wide association of SUPT16H, which may provide clue how SUPT16H facilitates HIV-1 latency. Since SUPT16H is a nucleosome chaperone protein, we will determine whether such activity of SUPT16H may alter for HIV-1 transcription. Our preliminary results suggest that depletion of SUPT16H further enhances a HDAC inhibitor to revert HIV-1 latency, so we will further investigate this result by broadly testing a set of HDACIs. AIM 3: Our earlier RNAi screens indicate that unbiased functional genomic tools are capable of systemically identifying previously unappreciated host proteins modulating HIV-1 replication. These screens also suggest that there are other potential LPGs pending for discovery. In this aim, we will use the newly emerged CRISPR/Cas9 functional genomic approach to systematically identify novel LPGs. We will perform comprehensive bioinformatic and biostatistic analyses to filter primary screening results and prioritize a set of the most promising LPGs (10~15 genes) for further validations in tonsillar CD4+ T cells, including confirmation of LPG depletion and re-evaluation of HIV latency reverting phenotype. Our ultimate goal is to translate this understanding to pharmacologically target a handful of LPGs for eradicating HIV-1 oral reservoirs.

 描述(申请人提供)：在HAART时代，HIV-1在感染者的储藏细胞中作为整合的潜伏前病毒持续存在，成为治愈的主要障碍。Tonsillr的CD4+T细胞是主要的HIV-1口腔储存库。了解这些细胞中调节HIV-1潜伏期的宿主因素并识别细胞特异性潜伏期促进基因(LPG)将有助于开发新的潜伏期反转剂(LRA)以消除HIV-1口腔蓄积物。我们早期的RNAi筛查成功地识别出限制HIV-1转录并促进其潜伏期的LPG，包括BRD4和SUPT16H。在这项建议中，我们建议在HIV-1潜伏感染的扁桃体CD4+T细胞(AIM 1和AIM 2)中进一步表征这两种LPG。它们对HIV-1潜伏期的贡献，特别是在口腔蓄积细胞中，在很大程度上是未知的，值得进一步描述。利用我们已建立的管道，我们还将有兴趣确定新的液化石油气(目标3)。我们相信我们建议的研究是全面的，并允许系统地观察LPG如何导致HIV-1潜伏期。目的1：我们是鉴定BRD4为LPG的主要小组之一，我们将进一步鉴定HIV-1潜伏感染的扁桃体CD4+T细胞中的BRD4。我们将首先使用原代扁桃体CD4+T细胞建立HIV-1潜伏期细胞模型。BRD4将被耗尽，以确定它如何影响HIV-1潜伏期/重新激活。我们的初步结果表明，P-TEFb介导了BRD4‘S效应，因此我们将进一步确定BRD4对扁桃体CD_4+T细胞中P-TEFb活性的影响。为了测试靶向BRD4以清除潜伏的HIV-1的可行性，我们将评估一组新的有希望的BET抑制剂来逆转HIV-1的潜伏时间。目的：SUPT16H是我们新发现的液化石油气，因此我们也将在HIV-1口腔储存库细胞中对其进行研究。我们将确定扁桃体CD4+T细胞中SUPT16H的耗尽是否会影响HIV-1潜伏期/重新激活。我们还将使用CHIP-SEQ方法来确定SUPT16H的全基因组关联，这可能为SUPT16H如何促进HIV-1潜伏提供线索。由于SUPT16H是一种核小体伴侣蛋白，我们将确定SUPT16H的这种活性是否会改变HIV-1的转录。我们的初步结果表明，SUPT16H的耗尽进一步增强了HDAC抑制剂以逆转HIV-1潜伏期，因此我们将通过广泛测试一组HDACI来进一步研究这一结果。目的3：我们早期的RNAi筛选表明，无偏见的功能基因组工具能够系统地识别先前未被认识的调节HIV-1复制的宿主蛋白。这些屏幕还表明，还有其他潜在的LPG有待发现。在这个目标中，我们将使用新出现的CRISPR/Cas9功能基因组学方法来系统地鉴定新的LPG。我们将进行全面的生物信息学和生物统计学分析，以筛选初步筛查结果，并优先选择一组最有希望的LPG(10~15个基因)，用于进一步验证扁桃体CD4+T细胞，包括确认LPG耗尽和重新评估HIV潜伏期恢复表型。我们的最终目标是将这一理解转化为药理学上针对少数LPG根除HIV-1口腔蓄积物。