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

 描述：潜伏的HIV-1感染已被认为是开发治愈的HIV-1疗法的主要障碍之一，然而，控制潜伏感染的细胞和病毒相互作用事件仍然定义不清。过去几年被宣传为艾滋病毒复活药物的HDAC抑制剂的使用引起了人们的认识，即可以而且应该检查根除艾滋病毒的情况。然而，HDAC抑制剂作为HIV-1复活剂的成功一直是有限的。这一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一种迫使病毒进入 潜伏状态，使细胞对刺激无反应。宿主T细胞的无能、无反应状态将最终解释尽管持续暴露于同源抗原，但迟发病毒库在体内的非凡稳定性。它还将解释最近的发现，这些发现描述了体外患者材料中的大多数潜伏感染事件甚至对PHA刺激没有反应。我们以前证明过，即使在高水平诱导的核因子-kB活性存在的情况下，也存在控制潜伏的HIV-1感染的守门人激酶功能。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，但在潜伏感染的T细胞中，整个通路的活动都发生了改变。Kinome图谱揭示了新的药物靶点，我们已经成功地将这些靶点用于改变T细胞系和潜伏HIV感染的初级T CLL模型中对潜伏HIV-1感染的控制。我们还提供了成功接受抗逆转录病毒治疗的HIV-1患者的CD4T细胞在运动学水平上发生了大规模改变的数据。虽然染色体的变化比T细胞系更复杂，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，并用于扰乱潜在HIV储存库的稳定性。鉴于这一观察结果，在这一应用中，我们建议使用HIV-1感染患者的体外T细胞材料来生成最相关的记忆T细胞亚群的更详细的基因组图谱，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将用于探测已识别的控制潜伏的HIV-1感染的目标，并开发有效触发HIV-1重新激活的新型药物组合。这项应用的目标是建立一个全面的潜伏HIV-1感染模型，该模型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，并利用这一知识来驱动药物重新定位努力，以确定将扭转宿主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触发HIV-1重新激活。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复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.