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

 描述（由申请人提供）：在HAART时代，HIV-1作为整合的潜伏前病毒持续存在于受感染个体的储库细胞中，是治愈的主要障碍。扁桃体CD 4 + T细胞是主要的HIV-1口腔储库。了解这些细胞中调节HIV-1潜伏期的宿主因素并鉴定细胞特异性潜伏期促进基因（LPGs）将有助于开发新的潜伏期逆转剂（LRAs）以消除HIV-1口腔储库。我们早期的RNAi筛选成功鉴定了限制HIV-1转录并促进其潜伏的LPG，包括BRD 4和SUPT 16 H。在这个提议中，我们建议进一步表征HIV-1潜伏感染的扁桃体CD 4 + T细胞（AIM 1和2）中的这两种LPG。它们对HIV-1潜伏期的贡献，特别是在口腔储库细胞中，在很大程度上是未知的，值得进一步表征。利用我们已建立的管道，我们也将有兴趣确定新的LPG（AIM 3）。我们相信我们提出的研究是全面的，并允许系统地了解LPGs如何促进HIV-1潜伏期。目标1：我们是将BRD 4鉴定为LPG的领导小组之一，我们将进一步表征HIV-1潜伏感染的扁桃体CD 4 + T细胞中的BRD 4。我们将首先使用原代扁桃体CD 4 + T细胞建立HIV-1潜伏细胞模型。BRD 4将被耗尽，以确定它如何影响HIV-1的潜伏期/再激活。我们的初步结果表明P-TEFb介导BRD 4的作用，因此我们将进一步确定BRD 4对扁桃体CD 4 + T细胞中P-TEFb活性的影响。为了测试靶向BRD 4抑制剂用于清除潜伏的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潜伏期。目的2：SUPT 16 H是我们新发现的LPG，因此我们也将在HIV-1口腔储库细胞中对其进行研究。我们将确定是否SUPT 16 H在扁桃体CD 4 + T细胞的耗竭可能会影响HIV-1的潜伏期/再激活。我们还将使用ChIP-seq方法来确定SUPT 16 H的全基因组关联，这可能提供SUPT 16 H如何促进HIV-1潜伏期的线索。由于SUPT 16 H是一种核小体伴侣蛋白，我们将确定SUPT 16 H的这种活性是否会改变HIV-1的转录。我们的初步结果表明，SUPT 16 H的耗尽进一步增强了HDAC抑制剂逆转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。目标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以前不受重视的调节HIV-1复制的宿主蛋白。这些筛选还表明，还有其他潜在的LPG有待发现。为此，我们将使用新出现的CRISPR/Cas9功能基因组方法来系统地鉴定新型LPGs。我们将进行全面的生物信息学和生物统计学分析，以筛选初步筛选结果，并优先考虑一组最有希望的LPG（10~15个基因），用于扁桃体CD 4 + T细胞的进一步验证，包括确认LPG耗尽和重新评估HIV潜伏期回复表型。我们的最终目标是将这一认识转化为针对少数LPGs根除HIV-1口腔储库。

 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.