Determining and Overcoming barriers in translating HIV-1 protease mediated activation of the CARD8 inflammasome to HIV cure research

项目摘要/摘要 自20世纪80年代S和38年以来，人类免疫缺陷病毒一直保持着全球流行的地位 全球有100万艾滋病毒携带者。虽然新的艾滋病毒感染人数一直在下降，抗逆转录病毒 治疗(ART)可以维持对PLWH的病毒抑制，目前还没有有效的治疗方法来消除病毒 从身体上。HIV擅长通过播种潜伏病毒来逃避正常的免疫反应 主要储存在静止的CD4T细胞中。一旦停止ART，这些潜在的储集层就能够 传播感染。除了这一障碍外，艾滋病毒还能够逃脱适应性免疫反应，因为 依赖于高度可变的艾滋病毒信封的识别。因此，制定一个治愈策略是至关重要的。 这可以感觉到艾滋病毒高度不变的方面，比如那些具有基本酶活性的病毒。我们以前的 研究表明，HIV-1蛋白酶是一种可以成为治愈策略的靶点的蛋白质。这种模式 识别受体Caspase募集结构域包含蛋白8(CARD8)能够感知HIV-1蛋白酶 并激活上睑下垂，一种炎症性的程序性细胞死亡。这一策略需要 通过使用非核苷可以实现的细胞内蛋白水解酶的过早激活 逆转录酶抑制剂(NNRTI)Efavirenz或利培韦林。当感染艾滋病毒的人增加NNRTIs时 细胞，CARD8激活炎症小体并诱导下垂。另外，我之前的工作表明， 抑制二肽基肽酶9(DPP9)负调控因子可增强NNRTI介导的信号转导 上睑下垂。然而，在发现这种清除病毒库的方法期间，我们发现了重要的 CARD8炎症体激活的变异可归因于病毒和宿主。 事实上，不同的病毒株、不同的CARD8亚型、不同的细胞类型、不同的CD4T细胞活化程度不同，上睑下垂的效率也不同 州和CD4T细胞捐赠者。因此，这项研究的中心假设是存在遗传变异 在病毒和宿主中都可以赋予对NNRTI诱导的HIV-1蛋白酶激活的抗性 CARD8炎症体及通过DPP9致敏的CARD8炎症体可被克服 抑制力。我将首先测试通过抑制DPP9来增强CARD8的临床和体内应用 目的1在人源化小鼠模型中测试临床分离株和增强作用。AIM 2中的实验将 确定CARD8检测HIV-1蛋白酶活性以告知基因组所需的关键病毒成分 对HIV-1进行广泛关联研究，以确定导致下睑下垂变异的变异。目标3将阐明 可能导致蛋白酶感知或炎症体激活变化的宿主因素。这一点将被确定 通过对HIV-1感染的关键细胞靶点异构体表达的研究，通过鉴定 通过潜在地识别CARD8的新的负调控因子，以不同的杀伤效率的捐赠者。 总的来说，这些研究将描述NNRTI诱导的CARD8炎症体的潜在疗效 激活作为一种减少病毒库的策略，是将这项工作转化为临床的第一步。

Project Summary/Abstract Human immunodeficiency virus (HIV) has maintained its status as a global epidemic since the 1980’s and 38 million people are living with HIV globally. While new HIV infections have been decreasing and antiretroviral therapies (ART) can maintain viral suppression in PLWH, there is no effective cure for elimination of the virus from the body. HIV is adept in escaping the normal immune response through the seeding of viral latent reservoirs primarily in quiescent CD4+ T cells. Upon halting ART these latent reservoirs are capable of spreading infection. In addition to this hurdle, HIV is able to escape the adaptive immune response due to the reliance upon recognition of the highly mutable HIV envelope. Therefore, it is critical to develop a cure strategy that can sense a highly immutable aspect of HIV such as those with essential enzymatic activity. Our previous work has shown that HIV-1 protease is one such protein that can be a target for a cure strategy. The pattern recognition receptor caspase recruitment domain-contain protein 8 (CARD8) is able to sense HIV-1 protease and activate pyroptosis, an inflammatory version of programmed cell death. This strategy requires the premature intracellular activation of the protease which can be achieved through the use of non-nucleoside reverse transcriptase inhibitors (NNRTIs) Efavirenz or Rilpivirine. Upon addition of NNRTIs to HIV infected cells, CARD8 activates the inflammasome and induces pyroptosis. Additionally, my previous work showed that inhibition of the negative regulator of dipeptidyl peptidase 9 (DPP9) is able to enhance NNRTI-mediated pyroptosis. However, during the discovery of this method for viral reservoir clearance, we found significant variation in the activation of the CARD8 inflammasome that could be ascribed to both the virus and the host. In fact, pyroptosis efficiency varied across viral strains, CARD8 isoforms, cell types, CD4+ T cell activation state, and CD4+ T cell donors. Therefore, the central hypothesis of this study is that there are genetic variants in both the virus and the host that can confer resistance to NNRTI induced HIV-1 protease activation of the CARD8 inflammasome and which may be overcome by CARD8 inflammasome sensitization through DPP9 inhibition. I will first test clinical and in vivo applications of CARD8 enhancement through DPP9 inhibition in Aim 1 by testing clinical isolates and enhancement in a humanized mouse model. Experiments in Aim 2 will identify the critical viral components needed for CARD8 sensing of HIV-1 protease activity to inform a genome wide association study of HIV-1 to identify variants responsible for pyroptosis variation. Aim 3 will elucidate host factors that may cause variation in protease sensing or inflammasome activation. This will be ascertained through the study of isoform expression in the key cellular targets for HIV-1 infection, through the identification of donors with varying killing efficiency, and through potentially identifying novel negative regulators of CARD8. Collectively, these studies will describe the potential efficacy of NNRTI induced CARD8 inflammasome activation as a viral reservoir reduction strategy and is the first step in translating this work to the clinic.