Novel Model for HIV Latency in Primary Memory T Cells

描述(由申请人提供)：在接受HAART治疗的患者中，HIV在记忆T细胞中长期处于潜伏状态，阻止了艾滋病毒的根除，并迫使患者终身服用HAART。虽然HIV的转录调控已经在转化的细胞系中进行了广泛的研究，但我们对潜在的HIV感染是如何发生在初级记忆的CD4T淋巴细胞中的理解还很初级。这项应用的目的是开发新的单细胞技术，以检测体外感染后单个初级淋巴细胞中HIV的转录状态。这些研究将架起人类免疫学和艾滋病毒分子病毒学两个研究领域的桥梁。了解HIV在初级淋巴细胞中的潜伏期可能会导致识别控制HIV潜伏期进入、潜伏期的维持或其重新激活的细胞蛋白。这种细胞靶标可成为治疗吸毒者艾滋病毒/艾滋病的新途径，并可能导致根除感染。我建议使用一种新的活细胞，时间推移荧光显微镜结合微流控芯片的细胞捕获，并使用表达HIV的重组荧光蛋白(失稳的GFP)在单细胞水平上研究HIV转录的动力学。这项新技术将允许随着时间的推移，在活的单个细胞中跟踪艾滋病毒表达的命运。人类淋巴细胞将在体外被激活，感染表达荧光蛋白的HIV，激活信号将被移除，随着时间的推移，HIV转录将被跟踪。我们预计，HIV转录将在恢复静止的一部分细胞中受到限制。在限制HIV转录激活的同时，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到HIV整合的关键。激活信号的性质也可能被证明是至关重要的。实验将最终实现

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 eventu