The role of chemokines in the establishment of HIV latency

描述(由申请人提供)：使用联合抗逆转录病毒疗法(ART)治疗艾滋病毒已显著降低了发病率和死亡率，但仅靠ART不能根除艾滋病毒感染。根除艾滋病毒的主要障碍是持久的、长期存活的、潜伏感染的静止的CD4+T细胞。制定封堵策略 除了抗逆转录病毒治疗外，建立潜伏感染还可以用来消除或加速潜伏感染的CD4+T细胞的衰变。我们的总体假设是，特定的趋化因子，即参与控制T细胞迁移和再循环的分子，对于促进 静息中的CD4+T细胞潜伏感染。我们的总体目标是确定新的干预措施，通过靶向宿主而不是病毒因素来阻断静止的CD4+T细胞的感染。为了解决这个问题，我们将 使用我们最近开发的稳健模型来建立体外潜伏期。在这个模型中，静息的CD4+T细胞与趋化因子一起孵育，趋化因子与静息细胞上高表达的趋化因子受体结合。在与相关趋化因子孵育后，细胞感染艾滋病毒并高度整合，没有观察到病毒产生，这与潜伏感染一致。利用这种趋化因子诱导潜伏期的模型，我们将首先确定趋化因子受体或趋化因子拮抗剂是否能抑制潜伏期的建立。趋化因子拮抗剂是N末端逐渐截断的趋化因子，它获得拮抗剂而不是激动剂的活性。我们将专门针对趋化因子受体，我们已经证明了这些受体对HIV感染静息的CD4+T细胞至关重要。其中包括CXCR3、CCR6和CCR7。趋化因子受体和趋化因子拮抗剂将在体外单独和联合进行评估。然后，我们将确定趋化因子如何在静止的CD4+T细胞中介导有效的核定位和整合的机制。我们将首先确定病毒迁移到静止的CD4+T细胞中的细胞核所需的关键细胞骨架通路。使用荧光显微镜，我们将确定关键通路是否涉及肌动蛋白或微管细胞骨架。我们将确定细胞骨架的变化是否有助于静止的CD4+T细胞的核定位，以及这是由特定的信号事件激活的，还是仅由T细胞迁移激活的。最后，我们将探讨磷脂酰肌醇-3-激酶(PI3K)通路在介导HIV在静止的CD4+T细胞中的有效整合中的作用。我们将确定对促进整合至关重要的相关下游蛋白质和特定的核因子。我们还将在这种潜伏期模型中定义HIV整合的位点，并确定整合和转录因子结合位点之间的关系。总体而言，这些研究可能最终确定新的抗病毒靶点，特别是针对 阻断静息CD4+T细胞的潜伏感染。

DESCRIPTION (provided by applicant): The use of combination antiretroviral therapy (ART) for the treatment of HIV has resulted in a significant reduction in morbidity and mortality, however HIV infection cannot be eradicated with ART alone. The major barrier to eradication of HIV is persistent long lived latently infected resting CD4+ T-cells. Developing strategies to block the establishment of latent infection could be used in addition to ART to eliminate or accelerate the decay of latently infected CD4+ T-cells. Our overall hypothesis is that specific chemokines, molecules involved in controlling T cell migration and recirculation, are critical for facilitating latent infection in resting CD4+ T-cells. Our overall aim is to identify novel interventions to blok infection of resting CD4+ T-cells by targeting host and not viral factors. To address this, we will use a robust model we have recently developed to establish latency in vitro. In this model, resting CD4+ T-cells are incubated with chemokines that bind to chemokine receptors highly expressed on resting cells. After incubation with the relevant chemokines, cells are infected with HIV and high levels of integration and no virus production is observed, consistent with latent infection. Using this model of chemokine-induced latency, we will first determine whether the establishment of latency can be inhibited by either chemokine receptor or chemokine antagonists. Chemokine antagonists are chemokines with progressive truncation of the N-terminus end which acquire antagonist rather than agonist activity. We will specifically target chemokine receptors that we have shown are critical for HIV infection of resting CD4+ T-cells. These include CXCR3, CCR6 and CCR7. The chemokine receptor and chemokine antagonists will be evaluated alone and in combination in vitro. We will then define the mechanisms of how chemokines mediate efficient nuclear localization and integration in resting CD4+ T-cells. We will first identify the critical cytoskeletal pathways required for migration of the virus to the nucleus in a resting CD4+ T-cell. Using fluorescent microscopy we will identify whether the key pathways involve the actin or microtubule cytoskeleton. We will identify whether changes in the cytoskeleton facilitate nuclear localization in resting CD4+ T-cells and whether this is activated by a specific signaling event or simply by T-cell migration alone. Finally, we will explore the rol of the phophoinosito-3-kinase (PI3K) pathway in mediating efficient HIV integration in resting CD4+ T-cells. We will determine the relevant downstream proteins and specific nuclear factors that are important for facilitating integration. We will also define the sites of HIV integration i this model of latency and determine the relationship between integration and transcription factor binding sites. Overall, these studies may ultimately identify novel antiviral targets, specific for blocking latent infection of resting CD4+ T-cells.