Cooperation of BRD4 and TAT Associated Proteins in HIV Transcription and Latency

 描述：虽然高效抗逆转录病毒疗法(HAART)成功地阻断了艾滋病患者中艾滋病毒的活跃复制，但它并不能根除病毒。潜伏的艾滋病毒宿主的存在仍然是治愈的主要障碍。从潜伏期重新激活病毒复制可引起细胞病变，并导致细胞死亡。目前有几种有希望的小化合物正在测试中，用于从潜伏期重新激活艾滋病毒。然而，它们要么在体内效力较弱，要么与严重毒性有关。最近发现，潜伏的艾滋病毒宿主的大小比之前估计的要大得多，这表明可能需要针对艾滋病毒潜伏的多种限制性机制的组合方案。因此，寻找新的基因靶点用于抗潜伏期治疗迫在眉睫。我们最近对调节HIV复制的宿主因素的研究发现，溴结构域蛋白BRD4在维持HIV潜伏期方面发挥着重要作用。更重要的是，我们发现BRD4抑制剂JQ1能够与其他HIV潜伏期逆转因子协同作用，在某些情况下激活潜伏的HIV。 这些初步数据表明，BRD4是一个有希望的HIV抗潜伏期治疗的基因靶点，但理想的基因与BRD4同时靶向以达到更好的效果仍有待确定。我们最近的研究结合了对与HIV Tat蛋白物理相关的宿主蛋白的蛋白质组学研究和利用RNAi介导的基因沉默调控HIV复制的宿主蛋白的遗传结果，发现了一组通过相互作用Tat来负调控HIV转录的HIV Tat相关抑制蛋白(TIPS)。在这项提案中，我们将首先验证一小组TIPS在HIV转录和原代CD4+T细胞潜伏期中的作用。我们将进一步研究它们与BRD4的合作互动。测量了TIPS和BRD4的遗传相互作用，以及在HIV潜伏期和重新激活时TIPS与TAT-LTR元件的物理相互作用。这些数据经过了对这个小型主机网络的计算分析，包括TAT-Ltr、BRD4-P-TEFb和TIPS。我们的目标是找出这个网络中的运动学和热力学上有意义的提示，这些提示可以成为小化合物的靶点。将为高通量小化合物筛选开发一种体外TIP-TAT相互作用试验，以确定可能破坏这种相互作用的抑制剂。这些化合物可能会释放TIP对TAT活性的抑制作用，从而充分激活HIV转录，从而有助于 恢复HIV潜伏期。我们将在HIV潜伏期的原代细胞模型以及从HAART治疗的艾滋病患者中分离的CD8+T细胞耗尽的外周血单个核细胞(PBMC)中对它们进行评估。还将确定这些化合物与BRD4抑制剂JQ1的有益效果，以确定使用JQ1进行HIV抗潜伏期治疗的更好药物对(S)。我们相信，我们对新TIPS的研究将提高我们对控制HIV转录和潜伏期的宿主机制的理解，并进一步针对那些可用于逆转HIV潜伏期和消除的药物。

 DESCRIPTION: Although highly active antiretroviral therapy (HAART) is successful to block active replication of HIV in AIDS patients, it does not eradicate viruses. Presence of latent HIV reservoirs remains a major obstacle to the cure. Reactivated viral replication from latency induces cytopathic effects and leads to cell death of reservoir cells. There are several promising small compounds being currently tested for HIV reactivation from latency. However, they are either less potent in vivo or associate with severe toxicity. Recently, it was uncovered that the size of latent HIV reservoirs is much larger than previously estimated, suggesting that a combinatory regimen targeting multiple restrictive mechanisms of HIV latency may be required. Thus, identification of novel gene targets for anti-latency therapy is urgent. Our recent studies o host factors modulating HIV replication identified the bromodomain protein, BRD4, plays an important role in maintaining HIV latency. More importantly, we found that the BRD4 inhibitor, JQ1, is able to synergize with other HIV latency reversers to activate latent HIV in certain cases. These preliminary data suggest that BRD4 is a promising gene target for HIV anti-latency therapy, but the ideal gene to be targeted simultaneously with BRD4 for reaching a better effect remains to be identified. Our recent studies, using an integrated approach to combine proteomic study of host proteins physically associating with HIV TAT protein and genetic results of host proteins regulating HIV replication using RNAi-mediated gene silencing, identified a set of HIV TAT-associated inhibitory proteins (TIPs) that negatively regulate HIV transcription through interacting TAT. In this proposal, we will first verify the role of a small set of TIPs in HIV transcription and latency in primary CD4+ T cells. We will further study their cooperative interactions with BRD4. Genetic interactions of TIPs and BRD4 as well as the physical interactions of TIPs with TAT-LTR elements at HIV latency and reactivation are measured. These data are subjected to computational analyses of this small host network including TAT-LTR, BRD4-P-TEFb, and TIPs. We aim to identify the kinetically and thermodynamically significant TIPs in this network that can be targeted by small compounds. An in vitro TIP-TAT interaction assay will be developed for high-throughput small compound screens to identify inhibitors that potently disrupt such interaction. These compounds may release the suppressive effect of TIP on TAT activity to allow full activation of HIV transcription, and thus are useful to revert HIV latency. We will evaluate them in both primary cell model of HIV latency as well as the CD8+ T cells depleted peripheral blood mononuclear cells (PBMCs) isolated from HAART-treated AIDS patients. Beneficial effect of these compounds together with BRD4 inhibitor JQ1 will be determined as well to identify better drug pair(s) for HIV anti-latency therapy using JQ1. We believe that our study of novel TIPs will improve our understanding of host machineries controlling HIV transcription and latency, and further target those druggable ones for reverting HIV latency and elimination.