HIV infection-induced mitochondrial dysfunction and premature T cell aging

HIV感染导致线粒体功能障碍和T细胞过早老化 HIV感染似乎导致T细胞过早衰老，线粒体功能障碍就是证据。CD4T细胞如何 在艾滋病毒感染过程中发生线粒体功能障碍的原因尚不清楚。这项建议的目的是澄清 探讨慢性HIV感染过程中线粒体功能障碍的机制，以开发有效的抢救手段 CD4T细胞耗尽或功能受损，是HIV感染的必备条件。以阐明其作用机制 在CD4T细胞衰老的线粒体功能障碍的基础上，我们分析了CD4T细胞的线粒体功能 来自抗逆转录病毒药物控制的艾滋病毒患者。我们的初步数据显示，HIV CD4T细胞已经减少 线粒体DNA(MtDNA)含量、线粒体呼吸和ATP产生。确定候选蛋白质 涉及线粒体DNA拷贝数失调，我们进行了液质联用(LC-MS) 从HIV患者和健康人(HS)的CD4T细胞中提纯线粒体。我们发现降幅最大的是 线粒体蛋白(SOD1和PRDX1)在破坏ROS和修复ROS中的作用 介导的DNA损伤修复(APEX1)和线粒体DNA降解中蛋白质的增加(exog和endog)以及 线粒体DNA复制(Polg和MGME1)。基于这些和其他初步数据，我们假设ROS- 介导的线粒体DNA损伤(通过较低的SOD1和/或PDRX1和APEX1)可能导致更高的mtDNA降解(通过 Exog和endog)，这可能不能得到mtDNA复制的充分补充(通过更高的Polg和 MGME1)，导致线粒体DNA拷贝数减少，线粒体功能受损，这是我们在HIV中看到的- 衍生的CD4T细胞。我们提出两个目标来定义导致线粒体dna减少和受损的机制。 功能。在目标1中，我们将确定SOD1和/或PRDX1的异位表达是否可以降低ROS水平和 HIV患者CD4T细胞线粒体DNA氧化损伤此外，还将进行APEX1的异位表达 探讨APEX1通过碱基切除修复(BER)途径修复线粒体DNA损伤的作用。 还将在健康的CD4T细胞中进行SOD1和/或PRDX1和APEX1的siRNA敲除以证实 它们在线粒体DNA损伤和拷贝数维持、线粒体呼吸和三磷酸腺苷生产中的作用。在AIM 2，我们将使用瞬时siRNA敲除或CRISER/Cas9敲除来减少外显和/或内切核酸酶 在HIV患者的CD4T细胞中，评估mtDNA氧化损伤水平和mtDNA拷贝的挽救 数。我们将在培养的CD4T细胞中对线粒体DNA进行复制DNA的单分子分析 以全面评估线粒体DNA复制状态对T细胞受体(TCR)的反应 刺激。总体而言，这项应用在概念和方法上都是新颖和强大的，以回答临床相关问题 问题：慢性病毒感染如何导致线粒体功能障碍，导致T细胞过早老化，以及 是否干扰那些导致线粒体DNA拷贝数减少和线粒体过度激活的酶 在HIV感染期间，功能障碍可以重塑T细胞的老化和功能。了解这些机制是至关重要的 用于开发在许多传染病或炎症性疾病中改善免疫反应的方法。

HIV infection-induced mitochondrial dysfunction and premature T cell aging HIV infection appears to drive premature T cell aging, evidenced by mitochondrial dysfunction. How CD4 T cells develop mitochondrial dysfunction during HIV infections is unclear. The objective of this proposal is to elucidate the mechanisms of mitochondrial dysfunction during chronic HIV infection, so as to develop effective means to rescue CD4 T cell depletion or functional impairment, the sine que non of HIV-infection. To elucidate the mechanisms underlying mitochondrial dysfunction in CD4 T cell aging, we analyzed the mitochondrial function of CD4 T cells derived from ART-controlled HIV patients. Our preliminary data show that HIV CD4 T cells have decreased mitochondrial DNA (mtDNA) content, mitochondrial respiration, and ATP production. To identify candidate proteins involved in dysregulating mtDNA copy numbers, we performed Liquid Chromatography Mass Spectrometry (LC-MS) on purified mitochondria from CD4 T cells of HIV patients and health subjects (HS). We found largest reduction of mitochondrial proteins (SOD1 and PRDX1) in destroying reactive oxygen species (ROS), and in repair of ROS- mediated DNA damage repair (APEX1), and elevation of proteins in mtDNA degrading (EXOG and ENDOG) and mtDNA replication (POLG and MGME1). Based on these and other preliminary data, we hypothesize that ROS- mediated mtDNA damage (via lower SOD1 and/or PDRX1 and APEX1) may cause higher mtDNA degradation (by EXOG and ENDOG), which may not be sufficiently complemented by mtDNA replication (through higher POLG and MGME1), leading to lower mtDNA copy number and impaired mitochondrial functions that we have seen in HIV- derived CD4 T cells. We propose two aims to define the mechanisms leading to mtDNA decrease and compromised function. In Aim 1, We will determine if ectopic expression of SOD1 and/or PRDX1 can reduce ROS level and oxidative mtDNA damage in CD4 T cells of HIV patients. In addition, ectopic expression of APEX1 will be performed to determine the involvement of APEX1 in repairing damaged mtDNA via the base excision repair (BER) pathway. siRNA knockdown of SOD1 and/or PRDX1, and APEX1 will also be performed in healthy CD4 T cells to confirm their roles in mtDNA damage and copy number maintenance, mitochondrial respiration, and ATP production. In Aim 2, we will use transient siRNA knockdown or Crisper/Cas9 knockout to reduce the EXOG and/or ENDOG nucleases in CD4 T cells from HIV patients, and to assess the levels of oxidative mtDNA damage and rescue of mtDNA copy number. We will perform single molecule analysis of replicated DNA (SMARD) on mtDNA in cultured CD4 T cells from HIV patients to comprehensively assess the status of mtDNA replication in response to T cell receptor (TCR) stimulation. Overall, this application is novel and strong in both concept and approach to answer clinically relevant questions: how chronic viral infection induces mitochondrial dysfunction, leading to premature T cells aging, and whether interfering those over-activated enzymes responsible for mtDNA copy number reduction and mitochondrial dysfunction can remodel T cell aging and function during HIV infection. Understanding such mechanisms is critical for developing approaches to improve immune responses in the setting of many infectious or inflammatory diseases.