Mechanisms underlying lncRNA SAF-mediated survival and persistence of HIV-1 infected macrophages

项目总结/摘要 联合抗逆转录病毒疗法在阻断HIV-1复制方面非常有效， 但是它不能消除在治疗开始之前感染的储库细胞。大多数HIV-1研究 持久性集中在潜伏感染的CD 4 + T细胞上。然而，组织驻留的巨噬细胞， 可自我更新的、长寿命的骨髓细胞，最近已显示在缺乏免疫原性的情况下维持长期的病毒血症。 T淋巴细胞。巨噬细胞支持HIV-1复制而不屈服于病毒的独特能力- 诱导的细胞死亡使它们成为病毒持久性的理想选择。了解病毒和宿主因素的作用， 使巨噬细胞逃避细胞死亡，需要制定有效的战略，消除这些 持续感染的骨髓细胞 通过筛选一组90个长链非编码RNA（lncRNA），我们最近发现了这种新的作用， lncRNASAF（FAS-AS 1）在维持HIV-1感染的巨噬细胞的细胞存活中的作用。这种lncRNA是 在HIV-1感染的人单核细胞衍生的巨噬细胞（MDM）中， 肺泡巨噬细胞（AM）。更重要的是，siRNA介导的SAF抑制导致SAF的激活。 在HIV-1感染的巨噬细胞中选择性地凋亡半胱天冬酶，而使病毒暴露但未感染 旁观者细胞不受影响。lncRNA SAF的这种高度特异性的调节导致细胞凋亡的显著减少。 巨噬细胞培养物中的病毒负荷，强调了靶向这种lncRNA的治疗潜力。我们 中心假设是，活动性HIV-1感染诱导巨噬细胞中SAF上调，以调节细胞凋亡。 存活/死亡途径促进病毒持久性。我们提出了一个深入的分析，既上游 SAF转录的调节子及其功能的下游效应子。 我们的具体目标是：（1）确定在HIV-1中诱导lncRNASAF的病毒和细胞调节剂 感染的巨噬细胞;（2）确定lncRNA SAF介导的保护巨噬细胞的分子机制。 HIV-1感染的巨噬细胞通过识别这种lncRNA的DNA/RNA和/或蛋白质靶点而免于细胞死亡。 我们将使用一组特定的复制阶段缺陷病毒， 和蛋白质组学工具（DNA/RNA-Seq，RNA反义纯化和LC-MS）来实现我们的目标。结果 这项研究将提高我们对HIV-1发病机制中lncRNA生物学的基本科学知识， 在巨噬细胞中的持久性。它还将通过确定 这种lncRNA的调节子和效应子。理解SAF介导的细胞存活的机制 并且HIV-1感染的巨噬细胞中的病毒持久性将极大地有助于最佳地开发新的治疗药物 这种lncRNA的潜力。

Project Summary / Abstract Combination anti-retroviral therapy is highly effective in blocking HIV-1 replication and thereby new infections but it cannot eliminate reservoir cells infected prior to the treatment initiation. Most studies of HIV-1 persistence have focused on latently infected CD4+ T cells. However, tissue-resident macrophages which are self-renewable, long-lived myeloid cells, have recently been shown to sustain prolonged viremia in absence of T lymphocytes. The unique ability of macrophages to support HIV-1 replication without succumbing to virus- induced cell death makes them ideal for viral persistence. Understanding the role of viral and host factors that enable macrophages to evade cell death is needed to develop effective strategies for eliminating these persistently infected myeloid cells. By screening a panel of 90 long non-coding RNAs (lncRNA), we have recently discovered the novel role of the lncRNA SAF (FAS-AS1) in maintenance of cell survival of HIV-1 infected macrophages. This lncRNA is significantly up-regulated in HIV-1 infected human monocyte-derived macrophages (MDM) in vitro and lung alveolar macrophages (AM) in vivo. More importantly, siRNA-mediated inhibition of SAF leads to activation of apoptotic caspases in HIV-1 infected macrophages selectively, while leaving the virus-exposed yet uninfected bystander cells unaffected. This highly specific modulation of the lncRNA SAF results in a marked reduction in viral burden in the macrophage culture, emphasizing the therapeutic potential of targeting this lncRNA. Our central hypothesis is that active HIV-1 infection induces SAF up-regulation in macrophages to regulate cell survival/death pathway(s) that promote viral persistence. We propose an in-depth analysis of both the upstream regulators of SAF transcription and the downstream effectors for its function. Our specific aims are to: (1) define the viral and cellular regulators for induction of lncRNA SAF in HIV-1 infected macrophages; and (2) determine the molecular mechanisms of lncRNA SAF mediated protection of HIV-1 infected macrophages from cell death by identifying the DNA/RNA and/or protein targets of this lncRNA. We will use a panel of specific replication-stage-defective viruses in combination with state-of-the-art genetic and proteomic tools (DNA/RNA-Seq, RNA antisense purification and LC-MS) to achieve our goals. The results from this study will improve our basic scientific knowledge of the biology of a lncRNA in HIV-1 pathogenesis and persistence in macrophages. It will also provide additional modalities for targeting SAF by identifying the regulators and effectors of this lncRNA. Understanding the mechanisms underpinning SAF-mediated cell survival and viral persistence in HIV-1 infected macrophages will aid greatly in optimally exploiting the novel therapeutic potential of this lncRNA.