Integrating subcellular multi-omics to identify druggable metabolic markers of latent HIV infection in CD4 T-cells

在被发现40年后，人类免疫缺陷病毒(HIV)继续感染全球近4000万人，每年造成数十万人死亡。艾滋病毒感染主要针对白细胞，如果不进行治疗，可能会导致严重的、可能致命的获得性免疫缺陷综合征(艾滋病)。抗病毒药物已经开发出来，并极大地提高了艾滋病毒携带者的预期寿命和质量。然而，这些药物需要终生服药，并不能治愈。重要的是，艾滋病毒在感染者的整个生命周期中以一种休眠(潜伏)的形式存在于一组白细胞(称为CD4T淋巴细胞)中。这一潜伏期使病毒能够躲避抗病毒药物和免疫系统。已经进行了几项研究，目的是确定持续感染细胞的特征，使我们能够专门针对这些细胞进行消除，从而有可能治愈感染。尽管已经提出了多种特征，但很少有安全治疗所需的特异性，而且大多数研究都未能减少持续感染细胞的频率。目前还没有针对持续感染艾滋病毒的细胞的治疗方法获得批准。正在进行临床测试的治疗方法之一是基于我们的研究表明，持续感染的细胞在能量代谢方面有特定的变化。然而，我们和其他小组的研究都集中在整个细胞上。事实上，大多数细胞能量的产生都位于高度专业化的亚细胞隔间内。为了使我们能够确定代谢调节的功能、结构和/或空间特征，以区分携带病毒的细胞和不携带病毒的细胞，拟议的项目将产生对持续感染艾滋病毒的细胞的深入表征(识别试剂盒)。我们将使用体外感染HIV的CD4T淋巴细胞和从HIV感染者的血液中分离出的CD4T淋巴细胞来完成这项工作。我们将首先根据感染的存在和阶段分离体外感染的细胞，然后分别研究主要的细胞能量产生和储存部位(即核/细胞质和线粒体)。获得的数据将全面捕捉蛋白质和代谢物的含量，以及支撑它们生产的基因调控。这些数据将通过计算结合起来，并由对每个主要细胞代谢步骤的功能的实验评估补充，以确定持续感染细胞的不同特征，这些特征可以探索其作为治疗靶点的潜力。然后，我们将使用计算机模型来预测有效的候选药物，并通过在实验室测试药物的靶向亲和力和选择性消除持续感染细胞的能力来实现这些预测。总体而言，这项研究的目的是提供艾滋病毒持久性的代谢决定因素的统一概况，并为消除对现有抗病毒药物具有耐药性的感染细胞的新治疗策略提供临床前证据。最终，这项工作可能导致一种治疗方法，将病毒从感染者的细胞藏身之处移除。

Four decades after its identification, the human immunodeficiency virus (HIV) continues to infect almost 40 million people worldwide, causing hundreds of thousands of deaths each year. Infection by HIV mainly targets white blood cells and, if left untreated, can lead to a severe, potentially fatal, acquired immunodeficiency syndrome (AIDS). Antiviral drugs have been developed and have drastically improved the life expectancy and quality of people living with HIV. However, these drugs require lifelong administration, and do not lead to a cure. Importantly, HIV persists in a dormant (latent) form in a subset of white blood cells (called CD4 T-lymphocytes) for the entire lifespan of infected individuals. This latency allows the virus to elude antiviral drugs and the immune system. Several studies have been conducted with the aim to identify features of persistently infected cells that could allow us to specifically target these cells for elimination and, thus, potentially cure the infection. Although multiple features have been proposed, few have the specificity required for safe therapeutic application, and most of the studies have failed to decrease the frequency of persistently infected cells. No current treatment to target persistently HIV-infected cells is approved. One of the therapeutic approaches undergoing clinical testing is based on our research showing that persistently infected cells have specific alterations in energy metabolism. Our studies, as well as those of other groups, however, focussed on whole cells. In reality, most cellular energy production is located within highly specialised subcellular compartments. To allow us to identify functional, structural, and/or spatial features of metabolic regulation that distinguish cells harbouring the virus from those that do not, the proposed project will produce an in-depth characterization (identikit) of persistently HIV-infected cells. We will approach this work both using CD4 T-lymphocytes infected with HIV in vitro and using CD4 T-lymphocytes isolated from the blood of individuals living with HIV. We will first separate cells infected in vitro based on the presence and stage of infection, and then individually study the main cellular sites of energy production and storage (i.e., nucleus/cytoplasm and mitochondria). The data obtained will comprehensively capture the content of proteins and metabolites, as well as the genetic regulation underpinning their production. These data will be computationally combined and complemented by experimental assessments of the functionality of each major cellular metabolic step in order to identify distinguishing features of persistently infected cells that can be explored for their potential to serve as therapeutic targets. We will then use computer models to predict effective drug candidates and put these predictions in practice by testing drugs in the laboratory for their target affinity and ability to selectively eliminate persistently infected cells. Overall, this study will aim to provide a unified profile of metabolic determinants of HIV persistence and furnish pre-clinical evidence for novel therapeutic strategies to eliminate infected cells resistant to currently available antiviral drugs. Ultimately, this work could lead to a therapeutic approach to remove the virus from its cellular hideout in infected individuals.