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（由适用提供）：在HAART时代，HIV-1在感染个体的储层细胞中持续存在，作为整合的潜在病毒，是治愈的主要障碍。扁桃体CD4+ T细胞是主要的HIV-1口腔储层。了解调节这些细胞中HIV-1潜伏期并鉴定细胞特异性潜伏基因（LPG）的宿主因子将对开发新的潜伏期逆转剂（LRA）有用，以消除HIV-1口服储层。我们较早的RNAi筛选成功鉴定了限制HIV-1转录并促进其潜伏期的LPG，包括BRD4和SUPT16H。在此提案中，我们建议进一步表征HIV-1潜在感染的扁桃体CD4+ T细胞中的这两个LPG（AIM 1和2）。它们对HIV-1潜伏期的贡献，尤其是在口腔储层细胞中，在很大程度上是未知的，值得进一步的表征。使用我们既定的管道，我们还将有兴趣识别NE LPG（AIM 3）。我们认为，我们提出的研究是全面的，并且可以系统地看待液化石油气如何促进HIV-1潜伏期。 AIM 1：我们是将BRD4识别为LPG的领先组之一，我们将进一步表征HIV-1潜在感染的扁桃体CD4+ T细胞中的BRD4。我们将首先使用主扁桃体CD4+ T细胞创建HIV-1潜伏细胞模型。 BRD4将被耗尽以确定其如何影响HIV-1潜伏/重新激活。我们的初步结果表明，P-TEFB介导了BRD4的效果，因此我们将进一步确定BRD4对扁桃体CD4+ T细胞中P-TEFB活性的影响。为了测试以清除潜在的HIV-1靶向BRD4药物的可行性，我们将评估一系列新的承诺BET抑制剂，以恢复HIV-1潜伏期。 AIM 2：SUPT16H是我们新鉴定的LPG，因此我们也将在HIV-1口腔储层细胞中进行研究。我们将确定在扁桃体CD4+ T细胞中SUPT16H的耗竭是否可能影响HIV-1潜伏/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，这可能提供SUPT16H如何准备HIV-1潜伏期的线索。由于SUPT16H是一种核小体链酮蛋白，因此我们将确定SUPT16H的这种活性是否可能改变HIV-1转录。我们的初步结果表明，SUPT16H的耗竭进一步增强了HDAC抑制剂以恢复HIV-1潜伏期，因此我们将通过广泛测试一组HDACIS进一步研究该结果。 AIM 3：我们较早的RNAi筛选表明，无偏的功能基因组工具能够系统地识别先前未经批准的宿主蛋白调节HIV-1复制。这些屏幕还表明，还有其他潜在的LPG待发现。在此目标中，我们将使用新出现的CRISPR/CAS9功能基因组方法系统地识别新型LPG。我们将进行全面的生物信息学和生物统计学分析，以过滤主要的筛选结果，并确定一组最有前途的LPG（10〜15基因），以在Tonsillar CD4+ T细胞中进行进一步验证，包括确认LPG定义和HIV延迟延迟降期振兴现象型。我们的最终目标是将这种理解转化为瞄准少数LPG，用于消除HIV-1口腔储藏。