Structural basis for cellular secretion and uptake of HIV-1 Tat

尽管抗逆转录病毒疗法在其他方面取得了成功， 约50%的感染者。大脑中HIV-1储库的存在被认为是导致 炎症环境和感染个体中的神经元损伤。在大脑中，HIV-1感染主要是由 由常驻的小胶质细胞和浸润的巨噬细胞组成ART在脑组织中的效率较低，因为 减少药物透过血脑屏障。此外，除了HIV-1感染的T细胞， 小胶质细胞/巨噬细胞屈服于病毒的细胞病变效应， 可以作为长期生产者。鉴于小胶质细胞的半衰期长，不同谱系的细胞具有不同的功能， 当巨噬细胞和可能的小胶质细胞增殖时，大脑本身就可能成为HIV-1感染的储存库 在HAND的背景下，即使ART完全抑制HIV-1复制， 感染的小胶质细胞/巨噬细胞可以主动分泌HIV-1转录反式激活因子（达特）蛋白， 对神经元尤其有害。已知达特与滥用药物协同作用， 疾病进展和严重程度，进一步混淆了对中枢神经系统（CNS）的不良反应。 越来越多的证据表明，HIV-1达特通过与人类多巴胺相互作用发挥其神经毒性 hDAT转运蛋白（hDAT）。最近也有证据表明，达特与淀粉样蛋白-β斑块相互作用， 阿尔茨海默氏病的标志，沉积在艾滋病毒感染患者的大脑中。达特分泌 作为一个有吸引力的目标，以改善手。然而，我们对达特是如何分泌的知识， 感染的细胞是有限的。相反，达特需要穿过靶细胞（如神经元）的膜来发挥其作用。 神经毒性作用因此，抑制摄取是改善HAND的第二个候选机制。 制备稳定的、有功能的重组全长达特是研究其分泌的障碍 和摄取机制，这也限制了药物发现测定的发展，以靶向“活性”形式 的达特来穿过细胞膜。为了实现这一应用，我们设计了新的方法来生成 大量的稳定和功能性达特，并采用核磁共振和生物物理工具，以确认其相互作用 用膜。我们还采用了功能测定来确认达特在神经元细胞培养中的功能。 基于我们的初步数据，我们假设达特在与 膜。我们的两个具体目标是：（i）鉴定达特在与 促进分泌的膜，和（ii）确定达特插入到 内体膜吸收。这里产生的结果将提供新的见解， 达特在分泌前和摄取过程中与膜结合时的分子重排， 一个新的平台，目标是膜结合形式的达特，这将导致新的发展， 用于治疗手的治疗策略。

Despite otherwise successful antiretroviral therapy, HIV-associated neurocognitive disorder (HAND) is detected in ~50% of infected individuals. The presence of an HIV-1 reservoir in the brain is considered a contributor to an inflammatory milieu and to the neuronal injury in infected individuals. In the brain, HIV-1 infection is driven mostly by resident microglia cells and infiltrated macrophages. ART is less efficient in the brain tissue because of reduced drug penetration through the blood-brain barrier. Moreover, other than HIV-1 infected T cells that succumb to the cytopathic effect of the virus, microglia/macrophages are resistant to the viral cytopathic effect and can act as long-term producers. Given the long half-life of microglia and the ability of different lineages of macrophages and likely microglia to proliferate, the brain presents itself as a likely reservoir of HIV-1 infection despite ART. In the context of HAND, even if ART were to completely suppress HIV-1 replication, persistently infected microglia/macrophages can actively secrete the HIV-1 transactivator of transcription (Tat) protein, which is known to be particularly toxic for neurons. Tat is known to synergize with drugs of abuse and exacerbate disease progression and severity, further confounding the adverse effects on the central nervous system (CNS). There is mounting evidence that HIV-1 Tat exerts its neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. There is also recent evidence that Tat interacts with amyloid-β plaques, a hallmark of Alzheimer's disease, that are deposited in the brain of HIV-infected patients. Tat secretion thus presents itself as an attractive target to improve HAND. However, our knowledge of how Tat is secreted by infected cells is limited. Conversely, Tat needs to cross membranes of target cells (such as neurons) to exert its neurotoxic effect. Therefore, inhibition of uptake is a second candidate mechanism to improve HAND. Preparation of a stable and functional recombinant full-length Tat has been a barrier to studying the secretion and uptake mechanisms, which also limits the development of drug discovery assays to target the “active” form of Tat needed to cross membranes. Leading to this application, we have devised new approaches to generate large amounts of stable and functional Tat and employed NMR and biophysical tools to confirm its interaction with membranes. We have also employed a functional assay to confirm Tat’s function in neuronal cell culture. Based on our preliminary data, we hypothesize that Tat undergoes a conformational change upon binding to membranes. Our two specific aims are: (i) identify the molecular rearrangements of Tat upon binding to membrane that facilitate secretion, and (ii) determine the molecular requirements for Tat insertion into endosomal membranes during uptake. The results generated here will provide novel insights into the molecular rearrangements of Tat upon binding to membranes prior to secretion and during uptake and will offer a new platform to target the membrane-bound form of Tat, which altogether will lead to the development of new therapeutic strategies for treatment of HAND.