Developing Pathogen Recognition Receptor Agonists as Latency Reversing Agents

潜伏的艾滋病毒感染细胞的存在构成了病毒传播的主要障碍。 根除。HIV-1潜伏的宿主规模很小，但寿命极长。潜伏感染与 病毒基因表达水平检测不到，而且似乎不会导致细胞病变。然而，在重新激活时， 潜伏病毒进入活跃的复制模式，在这种模式下，它们完全有能力传播和诱导 疾病。目前该领域的想法是，假想药物的组合将重新激活潜伏期 病毒(潜伏期反转剂或LRA)与目前的抗逆转录病毒药物一起使用将是一种有效的方法 朝着根除病毒的方向前进。我们先前已经发现，TLR-1/2激动剂Pam3CSK4可以重新激活潜伏期 HIV-1。基于这一早期发现，我们建议进一步研究发展病原体的可能性。 识别受体(PRR)激动剂作为重新激活/根除潜伏的HIV-1感染的新策略。 在目标1中，我们计划表征多个PRR激动剂重新激活潜伏的HIV-1的能力。多PRR 激动剂是由InvivoGen开发的合成TLR激动剂，用作癌症治疗的辅助剂， 传染病和疫苗接种。我们使用肿瘤细胞系JLat的初步数据，这是一种主要的 HIV-1潜伏期(培养的中医模型)和从无氧血症患者分离的细胞表明，这些配体 有能力重新激活潜伏的HIV-1。此外，在这一目标中，我们还计划评估这一潜力 激动剂，以减少体外潜伏库。我们已经发现，GTP酶抑制剂DATHORE具有 重新激活潜伏的HIV-1病毒。已有研究提出，DATORE激活PRR线粒体抗病毒信号 蛋白质(MAV)。我们进一步证实了这一结果。基于这一概念，这一目标旨在 表征RIG-I/MDA-5/MAVS通路的操作是否具有治疗意义 朝着根除艾滋病毒-1的目标迈进。首先，我们将研究RIG-1和MDA-5是否通过 MAV可以重新激活潜伏的HIV-1。其次，已经证明HIV-1蛋白水解酶可以诱导溶酶体 RIG-I的降解，以避免感应和诱导干扰素反应。我们计划将其描述为 该机制是否参与了延迟的建立。最后，确定新的机制 参与潜伏期的维持将导致新的治疗方法的设计。我们有 进行了一项筛选，以确定与合成的TLR-2配体CL572协同的表观遗传调节剂。 我们已经确定了几条与CL572协同的途径。在目标3中，我们计划详细描述 PRR激动剂与HDACs和溴域蛋白这两个已知途径之间的协同作用机制， 和两个新的途径，O6-甲基鸟嘌呤-DNA甲基转移酶和sirtuin-1和-2。 这项提案的总体目标是推进可用于治疗的新靶点。 要么根除HIV-1感染的病毒库，要么在不治疗的情况下导致长期缓解 (功能治愈)。

The existence of latent reservoirs of HIV-infected cells constitutes the major impediment towards viral eradication. HIV-1 latent reservoirs are small, but extremely long-lived. Latent infection is associated with undetectable levels of viral gene expression and appears to be non-cytopathic. However, upon reactivation, latent viruses enter an active mode of replication in which they are fully competent for spread and induction of disease. The current thinking in the field is that a combination of hypothetical drugs that will reactivate latent viruses (Latency Reversing Agent or LRA), with present-day antiretroviral drugs, will be an effective approach toward viral eradication. We have previously found that Pam3CSK4, a TLR-1/2 agonist, can reactivate latent HIV-1. Based on this earlier finding, we propose to further investigate the possibility of developing Pathogen Recognition Receptor (PRR) agonists as novel strategies to reactivate/eradicate latent HIV-1 infection. In Aim 1, we plan to characterize the ability of multi-PRR agonists to reactivate latent HIV-1. Multi-PRR agonists are synthetic TLR agonists developed by InvivoGen to be used as adjuvants in cancer therapy, infectious diseases and vaccination. Our preliminary data using the tumoral cell line JLat, a primary model of HIV-1 latency (Cultured TCM model) and cells isolated from aviremic patients demonstrate that these ligands have the ability to reactivate latent HIV-1. Furthermore, in this aim we also plan to evaluate the potential of this agonist to reduce the latent reservoir in vitro. We have found that dynasore, a GTPase inhibitor, has the ability to reactivate latent HIV-1. It has been proposed that dynasore activates the PRR mitochondria antiviral signal protein (MAVS). We have further corroborated this result. Based on this notion, this aim is designed to characterize whether manipulation of the RIG-I/MDA-5/MAVS pathway can have therapeutic implications towards HIV-1 eradication. First, we are going to study whether RIG-1 and MDA-5 ligands that signal through MAVS can reactivate latent HIV-1. Second, it has been shown that the HIV-1 protease can induce lysosomal degradation of RIG-I to avoid sensing and the induction of an interferon response. We plan to characterize whether this mechanism is involved in the establishment of latency. Lastly, identifying novel mechanisms that are involved in the maintenance of latency would lead to the design of novel therapeutic approaches. We have performed a screening to identify epigenetic modulators that synergize with the synthetic TLR-2 ligand CL572. We have identified several pathways that synergize with CL572. In Aim 3, we plan to characterize in detail the mechanism of synergy between PRR agonists and two known pathways, HDACs and bromodomain proteins, and two novel pathways, O6-Methylguanine-DNA methyltransferase and sirtuin-1 and -2. The overall goal of this proposal is to move forward novel targets that can be exploited therapeutically to either eradicate viral reservoirs for HIV-1 infections or to induce a long-term remission without treatment (functional cure).