Understand the role of CARD8 inflammasome in HIV-1 infection

摘要 尽管有抗逆转录病毒疗法(Art)，但由于病毒的存在，hiv-1感染是无法治愈的。 潜伏性蓄水池主要存在于长寿的静息记忆中的CD4+T细胞。病毒潜伏的储备库 是根除艾滋病毒-1的主要障碍。根除HIV-1的“震慑与杀戮”战略 涉及使用潜伏期反转剂(LRA)来诱导病毒基因表达，这 使受感染的细胞容易受到病毒的细胞病变或免疫清除的影响。然而， 在接受抗逆转录病毒治疗的患者中，潜伏在细胞中的艾滋病毒-1对病毒或免疫媒介具有抵抗力。 细胞凋亡性死亡，通常携带突变以逃避T细胞或抗体的识别。 因此，针对病毒不变成分的新方法，如基本病毒 蛋白质的功能是必需的。炎性小体是一种关键的分子复合体，它介导 微生物或危险信号引起的炎症和嗜热性细胞死亡。在人类中， CARD8炎症体的生理配体(S)仍不清楚。我们的研究表明 HIV-1蛋白酶降解CARD8 N-末端结构域并释放C-末端 炎性小体激活。在HIV-1感染细胞中，病毒蛋白水解酶作为一个亚单位保持不活跃 病毒Gag-Pol多蛋白。病毒萌发后，通过Gag-Pol二聚化被激活。我们 表明非核苷逆转对细胞内HIV-1蛋白酶的过早激活 转录酶抑制物(NNRTI)触发对感染HIV的巨噬细胞CARD8的感知和杀伤 和CD4+T细胞。尽管存在大量病毒，但CARD8可以感觉到HIV-1的所有亚型 多样性。我们的发现表明，靶向激活CARD8炎症体是一种很有前途的方法 消除HIV-1潜伏宿主的战略。在这项拟议的研究中，我们将：1)进行体外实验 基于CARD8的潜在清除“震杀策略”评估与优化 HIV-1在患者CD4+T细胞中的表达；2)了解HIV-1蛋白酶的分子机制-- CARD8介导的炎性小体激活；3)在 人源化小鼠HIV-1潜伏期模型。我们提出的研究将促进对 CARD8炎症小体激活的生理机制及其在HIV-1中的作用 感染，这将为艾滋病毒治愈研究提供关键影响。

Abstract Despite antiretroviral therapy (ART), HIV-1 infection is not curable due to the presence of viral latent reservoirs primarily in long-lived resting memory CD4+ T cells. The viral latent reservoirs are the major barrier to HIV-1 eradication. The “shock and kill” strategy for HIV-1 eradication involves the use of latency-reversing agents (LRAs) to induce viral gene expression, which renders the infected cells susceptible to viral cytopathic effects or immune clearance. However, in ART-treated patients, the latent HIV-1 resides in cells is resistant to viral- or immune-mediated apoptotic cell death and often carries mutations to escape recognition by T cells or antibodies. Therefore, novel approaches to target immutable components of the virus such as essential viral protein functions are needed. Inflammasome is a critical molecular complex that mediates inflammation and pyroptotic cell death in response to microbial or danger signals. In humans, the physiologic ligand(s) for the CARD8 inflammasome remains unknown. Our studies demonstrate that HIV-1 protease degrades the CARD8 N-terminal domain and releases the C-terminus for inflammasome activation. In HIV-1-infected cells, the viral protease remains inactive as a subunit of viral Gag-Pol polyprotein. After virus budding, it is activated through Gag-Pol dimerization. We show that premature activation of intracellular HIV-1 protease by non-nucleoside reverse transcriptase inhibitors (NNRTI) triggers CARD8 sensing and killing of HIV-infected macrophages and CD4+ T cells. All subtypes of HIV-1 can be sensed by CARD8 despite substantial viral diversity. Our finding suggests that targeted activation of CARD8 inflammasome is a promising strategy to eliminate latent HIV-1 reservoirs. In this proposed study, we will: 1) perform ex vivo assessment and optimization of the CARD8-based “shock and kill strategy” for clearing latent HIV-1 in patient CD4+ T cells; 2) understand the molecular mechanisms of HIV-1 protease- mediated CARD8 inflammasome activation; 3) test CARD8-based “shock and kill” strategy in humanized mouse model of HIV-1 latency. Our proposed study will advance the understanding of the physiological mechanisms of CARD8 inflammasome activation and its role in HIV-1 infection, which will provide critical implications to HIV cure research.