Novel Model for HIV Latency in Primary Memory T Cells

描述(申请人提供)：接受HAART治疗的患者中HIV在记忆T细胞中潜伏状态的长期持续 阻止艾滋病毒的根除，迫使患者终生接受HAART治疗。而当 HIV的转录调控在转化的细胞系中得到了广泛的研究，我们对 潜伏的HIV感染是如何发生在初级记忆的CD4T淋巴细胞中的，目前尚不清楚。 这项应用的目的是开发新的单细胞技术来检测转录 体外感染后单个初级淋巴细胞中HIV的状态。这些研究将在 人类免疫学和HIV分子病毒学两个研究领域。了解HIV在小学的潜伏期 淋巴细胞可能导致识别控制HIV潜伏进入的细胞蛋白，即 潜伏期的维持或重新激活。这样的细胞靶点可能是治疗的新途径。 在吸毒者中传播艾滋病毒/艾滋病，并可能导致根除感染。 我建议使用一种新的活细胞，延时荧光显微镜和细胞捕获通过 微流控芯片与利用HIV表达重组荧光蛋白(失稳GFP)的研究 HIV在单细胞水平上的转录动力学。这项新技术将使艾滋病毒的命运 随着时间的推移，在活的单个细胞中遵循的表达。人类淋巴样细胞将在体外被激活， 感染了表达荧光蛋白的HIV，激活信号会被移除，HIV会被转录 将随着时间的推移而被追随。我们预计HIV转录将被限制在返回到 宁静。将激活信号的移除与感染分开的时间很可能是允许 感染继续进行，直到艾滋病毒整合，同时限制艾滋病毒转录激活。这个世界的本质 激活信号也可能被证明是关键的。实验最终将集中在高度浓缩的人类淋巴 细胞(幼稚vs.记忆力)和使用R5 HIV信封来密切模拟观察到的HIV感染情况 并开发一种接近于患者情况的体外模型。 当HIV潜伏期的体外模型建立后，我将研究FOXO3a的作用 转录因子。我们的假设是FOXO3a代表记忆T中HIV潜伏期的主要调节者 细胞。FOXO3a对记忆T细胞的维持和存活至关重要，强烈抑制NF-κB和IS 因此可能抑制艾滋病毒的表达，并有助于潜伏期的建立或维持。我们会 FOXO3a的PI3K/AKT细胞激活途径及SIRT1和SIRT3对FOXO3a的影响 功能和HIV潜伏期。最后，我们将研究最近在FOXO3a中发现的多态的影响 影响其功能的基因对HIV感染者潜伏池的大小和建立 在体外的潜伏期。

DESCRIPTION (provided by applicant): The long-term persistence of HIV in a latent state in memory T cells in patients treated with HAART prevents the eradication of HIV and forces patients to remain on HAART for their whole life. While the transcriptional regulation of HIV has been extensively studied in transformed cell lines, our understanding of how latent HIV infection occurs in primary memory CD4 T lymphocytes is rudimentary. The purpose of this application is to develop new single cell technology to examine the transcriptional status of HIV in single primary lymphoid cells over time after an infection in vitro. These studies will bridge the two research fields of human immunology and HIV molecular virology. Understanding HIV latency in primary lymphocytes may lead to the identification of cellular proteins that control the entry of HIV in latency, the maintenance of latency or its reactivation. Such cellular targets could represent new avenues for the treatment of HIV/AIDS among drug abusers and possibly lead to the eradication of infection. I propose to use a novel live cell, time-lapse fluorescence microscopy combined with cell trapping via microfluidic chips and the use of HIV expressing recombinant fluorescent protein (destabilized GFP) to study the kinetics of HIV transcription at the single cell level. This novel technique will allow the fate of HIV expression to be followed in live individual cells over time. Human lymphoid cells will be activated in vitro, infected with an HIV expressing a fluorescent protein, activation signals will be removed and HIV transcription will be followed over time. We anticipate that HIV transcription will be restricted in a subset of cells returning to quiescence. The time separating removal of activation signal to infection is likely to be critical in allowing infection to proceed until HIV integration while restricting HIV transcriptional activation. The nature of the activation signal could also prove critical. Experiment will eventually focus on highly enriched human lymphoid cells (naïve vs. memory) and on the use of R5 HIV envelope to closely mimic the situation observed in HIVinfected patients and to develop an in vitro model that closely mimics the situation in patients. When an in vitro model for HIV latency has been established, I will study the role of the FOXO3A transcription factor. Our hypothesis is that FOXO3A represents a master regulator of HIV latency in memory T cells. FOXO3A is critical for memory T cell maintenance and survival, strongly represses NF-κB and is therefore likely to repress HIV expression and to contribute to latency establishment or maintenance. We will study the PI3K/AKT cellular activation pathway for FOXO3A and the effect of SIRT1 and SIRT3 on FOXO3A function and HIV latency. Finally, we will study the effect of recently identified polymorphisms in the FOXO3A gene that affect its function on the size of the latent pool in patients infected with HIV and on the establishment of latency in vitro.