Novel Model for HIV Latency in Primary Memory T Cells

描述（由申请人提供）：接受HAART治疗的患者记忆T细胞中潜伏状态的HIV长期持续存在 阻止了艾滋病毒的根除，迫使患者终生接受高效抗逆转录病毒治疗。而 HIV的转录调控已经在转化细胞系中进行了广泛的研究，我们对 潜伏的HIV感染如何发生在初级记忆CD 4 T淋巴细胞中是基本的。 本申请的目的是开发新的单细胞技术来检查转录 体外感染后单个初级淋巴细胞中HIV的状态随时间的变化。这些研究将连接 人类免疫学和HIV分子病毒学两大研究领域。了解艾滋病毒在初级 淋巴细胞可能导致识别控制HIV潜伏期进入的细胞蛋白， 保持潜伏期或其重新激活。这些细胞靶点可能代表治疗的新途径 在吸毒者中预防艾滋病毒/艾滋病，并可能导致根除感染。 我建议使用一种新的活细胞，延时荧光显微镜结合细胞捕获， 微流控芯片和使用表达HIV的重组荧光蛋白（不稳定的GFP）来研究 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 在活的个体细胞中随时间的推移进行表达。人类淋巴细胞将在体外被激活， 感染了表达荧光蛋白的HIV，激活信号将被移除，HIV转录 将随着时间的推移而被跟踪。我们预计，艾滋病毒的转录将被限制在一个子集的细胞返回到 安静激活信号的去除与感染之间的时间间隔可能是关键， 感染进行，直到艾滋病毒整合，同时限制艾滋病毒转录激活。的性质 激活信号也是至关重要的。实验最终将集中在高度富集的人类淋巴细胞 细胞（幼稚与记忆）和使用R5 HIV包膜密切模拟HIV感染者中观察到的情况 患者，并开发一个体外模型，密切模仿患者的情况。 当HIV潜伏期的体外模型建立后，我将研究FOXO 3A的作用。 转录因子我们的假设是FOXO 3A代表了记忆T细胞中HIV潜伏期的主要调节因子。 细胞FOXO 3 A对记忆T细胞的维持和存活至关重要，强烈抑制NF-κB， 因此可能抑制HIV表达并有助于潜伏期的建立或维持。我们将 研究FOXO 3A的PI 3 K/AKT细胞活化途径以及SIRT 1和SIRT 3对FOXO 3A的作用 功能和HIV潜伏期。最后，我们将研究最近发现的FOXO 3A多态性的影响， 基因，影响其功能的大小潜伏池感染艾滋病毒的患者和建立 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.