Kinomic analysis of host cell factors controlling latent HIV-1 infection

 产品说明：潜伏性HIV-1感染已被认为是开发治愈性HIV-1疗法的一个主要障碍，然而，控制潜伏性感染的细胞和病毒相互作用事件仍然不明确。在过去几年中，HDAC抑制剂被推广为HIV再激活药物，这使人们意识到可以而且应该检查HIV根除。然而，HDAC抑制剂作为HIV-1再活化剂的成功是有限的。这种应用的关键是认识到潜伏的HIV-1感染事件的宿主细胞实际上是以迫使病毒进入一种非免疫状态的方式表型改变的。 潜伏状态并使细胞对刺激无反应。宿主T细胞的无能量样、无反应状态将最终解释latnt病毒储库的非凡体内稳定性，尽管持续暴露于同源抗原。这也解释了最近的研究结果，即离体患者材料中的大多数潜伏感染事件甚至对PHA刺激无反应。我们先前证明了即使在高水平诱导的NF-kB活性存在下，也存在控制潜伏的HIV-1感染的看门人激酶功能。我们的数据现在表明，宿主细胞的表型变化超出了单个激酶的功能，但整个途径的活动在潜伏感染的T细胞中发生了改变。激酶组分析揭示了新的药物靶点，我们已经成功地靶向这些靶点，以改变T细胞系和潜伏性HIV感染的原代T细胞II模型中潜伏性HIV-1感染的控制。我们还提供了来自成功ART的HIV-1患者的CD 4 T细胞在kinomic水平上被大量改变的数据。虽然kinomic的变化比T细胞系中更复杂，但受影响的T细胞信号通路完全重叠，这一发现可能解释了为什么在T细胞系中获得的一些发现可以直接转移到原代T细胞模型中，并用于干扰潜伏HIV库的稳定性。鉴于这一观察结果，在本申请中，我们提出使用来自HIV-1感染患者的离体T细胞材料生成已知宿主潜伏HIV-1感染事件的最相关记忆T细胞亚群的越来越详细的激酶组谱，并生成描述HIV-1潜伏期控制的蛋白质-蛋白质相互作用网络（PIN）。然后，生成的PIN将用于探测控制潜伏HIV- 1感染的已识别靶标，并开发有效触发HIV-1再激活的新型药物组合。本申请的目标是建立潜伏HIV-1感染的综合模型，其考虑病毒与宿主细胞在激酶、转录因子和可能的染色质水平上的动态双向相互作用，并使用该知识来驱动药物重新定位努力以鉴定将逆转宿主T细胞的无反应状态的药物组合，从而 允许同源抗原和可能的治疗刺激物触发HIV-1再活化。从所描述的研究中获得的见解将为如何基于未探索的分子药物靶点开发有效的HIV-1再激活治疗策略提供蓝图。

 DESCRIPTION: Latent HIV-1 infection has been recognized as one major obstacle to the development of a curative HIV-1 therapy, however, cellular and viral interaction events that control latent infection are still ill defined. The use of HDAC inhibitors that have been promoted as HIV reactivating drugs in the last years has generated awareness that HIV eradication can and should be examined. However, the success of HDAC inhibitors as HIV-1 reactivating agents has been limited. Key to this application is the realization that the host-cells of latent HIV-1 infection events are actually phenotypically altered in a manner that forces the virus into a latent state and renders the cells unresponsive to stimulation. An anergy-like, unresponsive state of the host T cells would conclusively explain the extraordinary in vivo stability of the latnt viral reservoir despite continuous exposure to cognate antigen. It would also explain recent findings describing that the majority of the latent infection events in ex vivo patient material ar even unresponsive to PHA stimulation. We previously demonstrated the presence of a gate-keeper kinase function that controls latent HIV-1 infection even in the presence of high levels of induced NF-kB activity. Our data now show that phenotypic changes of the host cells go beyond the functionality of individual kinases, but that the activities of entire pathways are altered in latently infected T cells. Kinome profiling revealed novel drug targets that we have already successfully targeted to alter control of latent HIV-1 infection in T cell lines and in primary T cll models of latent HIV infection. We also provide data that CD4 T cells from HIV-1 patients on successful ART are massively altered at the kinomic level. While the kinomic changes are more complex than in T cell lines, there is complete overlap in the affected T cell signaling pathways, a finding that likely explains why some of the findings obtained in T cell lines can be directly transferred into primary T cell models and used to perturb the stability of the latent HIV reservoir. Given this observation, in this application, we propose to generate increasingly detailed kinome profiles of the most relevant memory T cell sub-populations know to host latent HIV-1 infection events using ex vivo T cell material from HIV-1 infected patients and to generate protein-protein interaction networks (PIN) that describe HIV-1 latency control. The generated PINs will then be used to probe identified targets that control latent HIV- 1 infection and to develop novel drug combinations that efficiently trigger HIV-1 reactivation. The goal of this application is to establish a comprehensive model of latent HIV-1 infection that considers the dynamic, bi-directional interactions of the virus with the host-cell at the kinase, transcription factor and possibly chromatin level and to use this knowledge to drive a drug repositioning effort to identify drug combinations that will reverse the unresponsive state of the host T cells, thereby allowing cognate antigen and possible therapeutic stimuli to trigger HIV-1 reactivation. The insights gained from the described studies will provide a blueprint for how to develop efficient HIV-1 reactivating therapeutic strategies based on unexplored molecular drug targets.