Cellular Metabolic Reprogramming and Virus Infectivity

项目摘要 本研究提出以下假设：1.提前停药后病毒会自发产生 ART来自粘膜组织中最早建立的“储库细胞”（项目1旨在识别）。 粘膜组织中最早建立的储库细胞的候选者（滤泡辅助性T（Tfh），中央淋巴细胞）， 记忆T（Tcm）和Th 17细胞）将各自离体研究。T细胞潜伏期/再激活模型已经 将采用文献中描述的方法，并开发新的模型， 结肠粘膜以及人扁桃体。前者旨在研究Th 17，后者Tfh，细胞作为潜在的 我们还将试验人源化DRAG小鼠作为模型， 女性生殖道（FRT）粘膜Tfh水库，并确定是否扁桃体外植体或 人源化小鼠优选用于进一步研究Tfh细胞相关机制和干预（在目的2中 和3）。粘膜SIV感染后第3天开始ART抑制的猕猴，持续6天 在停止前几个月，还将与项目1密切合作进行研究。的观测研究 将设计和实施暂停>1年的抑制性ART之前和之后的HIV感染受试者 （“暂停ART后反弹的临床观察”，CORA）。2. mTOR介导的代谢重编程 由T细胞活化引发的病毒血症导致ART后的病毒血症反弹。 将使用催化mTOR抑制剂在离体组织和动物模型中研究mTOR活性，包括在 SIV感染的猕猴在停止ART前被ART抑制6个月。 从早期ART停止后的储库细胞，和靶细胞对病毒的易感性，将被表征。3.有限 或在停止早期ART后最初产生的病毒体中缺乏病毒体A3 G有助于致病级联反应 病毒血症反弹模型系统（离体和/或人源化DRAG小鼠）将用于测试病毒是否 储库T细胞甚至在Vif表达之前具有低A3 G水平（有助于病毒感染性），并且如果A3 G- 增强工具化合物降低了由T细胞库产生的病毒体的感染性。我们将评估A3 G- 介导的病毒传播减少增加了mTOR介导的未感染靶T细胞减少 感染的易感性，以及如果由mTOR抑制引起的A3 G细胞质分布的变化增强 A3 G增强剂介导的A3 G病毒粒子包装的增加。

Project Summary This project addresses the following hypotheses: 1. Virus will be spontaneously produced after stopping early ART from the earliest-established “reservoir cells” in mucosal tissues (which Project 1 aims to identify). Candidates for the earliest-established reservoir cells in mucosal tissues (follicular helper T (Tfh), central memory T (Tcm), and Th17 cells) will each be studied ex vivo. T cell latency/reactivation models already described in the literature will be adapted and new models developed that use explanted human and macaque colon mucosa, as well as human tonsils. The former aims to study Th17, and the latter Tfh, cells as potential early reservoirs that may initiate rebound after ART. We will also pilot humanized DRAG mice as a model of female reproductive tract (FRT) mucosal Tfh reservoirs, and determine whether the tonsil explants or humanized mice are preferable for further study of Tfh cell-related mechanisms and interventions (in aims 2 and 3). Macaques with ART-suppression starting at day 3 after mucosal SIV infection, and continuing for 6 months before stopping, will also be studied in close collaboration with project 1. An observational study of HIV-infected subjects before and after pausing >1 year-long suppressive ART will be designed and performed (“Clinical Observation of Rebound after pausing ART”, CORA). 2. mTOR-mediated metabolic reprogramming triggered by T cell activation contributes to viremia rebound off-ART. Cellular mechanisms downstream of mTOR activity will be studied using catalytic mTOR inhibitors in ex vivo tissue and animal models, including in SIV-infected macaques suppressed by ART for 6 months before stopping it. Effects on both virus production from reservoir cells after early ART stops, and target cell susceptibility to virus, will be characterized. 3. Limited or absent virion A3G in virions initially-produced after stopping early ART contribute to the pathogenic cascade to viremia rebound. Model systems (ex vivo and/or humanized DRAG mice) will be used to test whether virus reservoir T cells have low A3G levels even before Vif is expressed (contributing to virus infectivity), and if A3G- boosting tool compounds decrease infectivity of virions produced from T cell reservoirs. We will assess if A3G- mediated decreased virus spread adds to mTOR inhibitor-mediated decreases in uninfected target T cell susceptibility to infection, and if changes in A3G's cytoplasmic distribution caused by mTOR inhibition enhance A3G booster-mediated increases in A3G virion packaging.