The role of purinergic receptor signaling in the establishment of HIV latency

这项申请提出了一个项目，以检查嘌呤能受体信号在艾滋病毒感染中的作用。特别是，将分析三磷酸腺苷和腺苷信号在艾滋病毒复制周期和病毒潜伏期建立中的作用。为了研究嘌呤能受体(腺苷为P1，腺苷为P2X和P2Y为ATP)在HIV进入、复制和潜伏时间中的作用，我们设计了体外感染带有野生型HIV(JRFL)和报告HIV病毒粒子(R7GEmC，pMM310)的健康人外周血单个核细胞的方法，并与特定的嘌呤能受体激动剂和拮抗剂相结合。(由国立卫生研究院分子识别科雅各布森教授提供)。为探讨嘌呤能受体信号在HIV感染临床过程中的作用，采用下一代测序技术对HIV感染者不同病程的单个分选的HIV感染者的CD4+T细胞进行全转录组分析，包括联合抗逆转录病毒治疗(CART)。杜埃克实验室已经建立了一种基于液滴的微流控细胞分选程序，用于鉴定和分离艾滋病毒感染细胞。外周血液、淋巴结和肠道粘膜的样本将通过Douek博士与加州大学旧金山分校(Steven Deek博士)、凯斯西储大学(Michael Lederman博士)、明尼苏达大学(Timothy Schacker博士)以及NIH诊所的研究人员持续合作获得。目前，可从100多名艾滋病毒感染者那里获得纵向样本。实验工作将在国家卫生研究院(马里兰州贝塞斯达)的国家过敏和传染病研究所(NIAID)的校园内进行，该研究所位于疫苗研究中心(VRC)人类免疫学部，由Daniel Douek医学博士、MRCP博士领导。由Daniel Douek领导的VRC基因组分析核心可以获得足够的S3设施和技术设备，用于处理和单分类艾滋病毒感染材料以及整个转录组分析。

This application proposes a project to examine the role of purinergic receptor signaling in HIV infection. In particular, the role of ATP and adenosine signaling in the HIV replicative cycle and in the establishment of viral latency will be analyzed. To investigate the role of purinergic receptors (P1 for adenosine, P2X and P2Y for ATP) in HIV entry, replication and latency, in vitro infection assays of healthy, human peripheral blood mononuclear cells with wildtype HIV (JRFL) and reporter HIV virions (R7GEmC, pMM310) are planned in combination with specific purinergic receptor agonists and antagonists. (Provided by Prof. Jacobson, Molecular Recognition Section, NIH). To investigate the role of purinergic receptor signaling in the clinical course of HIV infection,whole transcriptome analysis using next generation sequencing will be carried out on single sorted, HIV-infected CD4+ T cells from HIV-infected people at different stages of disease, both on and off combination anti-retroviral therapy (cART). A droplet-based microfluidic cell sorting procedure for the identification and isolation of HIV-infected cells is already established in the Douek Laboratory. Samples of peripheral blood, lymph node and gut mucosa will be obtained through Dr. Doueks ongoing collaborations with investigators at the University of California San Francisco (Dr. Steven Deeks), Case Western Reserve University (Dr. Michael Lederman), The University of Minnesota (Dr. Timothy Schacker) and through the NIH clinic. Currently, longitudinal samples are available from over 100 HIV-infected individuals. The experimental work will be conducted at the National Institute of Allergy and Infectious Diseases (NIAID) at the campus of the National Institutes of Health (Bethesda, Maryland) in the Human Immunology Section of the Vaccine Research Center (VRC), headed by Daniel Douek M.D., MRCP, Ph.D. Adequate S3 facilities and technical equipment for handling and single-sorting of HIV infected materials and whole transcriptome analysis are accessible in the Genome Analysis Core of the VRC, headed by Daniel Douek.