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

项目总结/摘要 自1980年代以来，人类免疫缺陷病毒（艾滋病毒）一直是全球流行病， 全球有100万人感染艾滋病毒。虽然新的艾滋病毒感染一直在减少， 抗逆转录病毒疗法（ART）可以维持病毒抑制在PLWH，没有有效的治疗消除病毒 从尸体上艾滋病病毒善于通过接种潜伏的病毒来逃避正常的免疫反应， 主要存在于静止的CD 4 + T细胞中。在停止ART后，这些潜在的储库能够 传播感染除了这一障碍，艾滋病毒能够逃避适应性免疫反应，由于 依赖于对高度可变的HIV包膜的识别。因此，制定治疗策略至关重要 它可以检测HIV的一个高度不变的方面，例如那些具有基本酶活性的方面。我们以前的 研究表明，HIV-1蛋白酶就是这样一种蛋白质，可以作为治疗策略的靶点。图案 识别受体半胱天冬酶募集结构域蛋白8（CARD 8）能够感知HIV-1蛋白酶 并激活细胞凋亡，一种程序性细胞死亡的炎症形式。这一战略要求 蛋白酶的过早胞内活化，这可以通过使用非核苷 逆转录酶抑制剂（NNRTI）依法韦仑或依匹韦林。在HIV感染者中加入NNRTI后， 细胞，CARD 8激活炎性小体并诱导细胞凋亡。此外，我以前的工作表明， 抑制二肽基肽酶9（DPP 9）的负调节因子能够增强NNRTI介导的 焦亡然而，在发现这种用于病毒储库清除的方法的过程中，我们发现了显著的 CARD 8炎性体的活化的变化，其可归因于病毒和宿主。 事实上，细胞凋亡效率在病毒株、CARD 8亚型、细胞类型、CD 4 + T细胞活化、 状态和CD 4 + T细胞供体。因此，本研究的中心假设是， 在病毒和宿主中，可以赋予对NNRTI诱导的HIV-1蛋白酶激活的抗性， CARD 8炎性小体，并且其可以通过DPP 9被CARD 8炎性小体致敏克服 抑制作用我将首先测试通过DPP 9抑制CARD 8增强的临床和体内应用， 目的1通过在人源化小鼠模型中测试临床分离株和增强。目标2的实验将 确定CARD 8检测HIV-1蛋白酶活性所需的关键病毒组分，以告知基因组 HIV-1的广泛相关性研究，以确定导致细胞凋亡变异的变异体。目标3将阐明 可能引起蛋白酶感应或炎性小体激活变化的宿主因素。这一点将得到确认 通过研究HIV-1感染的关键细胞靶点中的同种型表达，通过鉴定 具有不同杀伤效率的供体，并通过潜在地鉴定CARD 8的新型负调节因子。 总的来说，这些研究将描述NNRTI诱导的CARD 8炎性小体的潜在疗效 激活作为病毒库减少策略，并且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.