Role of heme oxygenase 2 in trafficking and regulation of myristoylated proteins

我们将探索一种新的宿主因子在逆转录病毒复制中的作用，我们发现血红素加氧酶2(HO-2)与N-末端的肉芽孢子蛋白结合，并调节它们向质膜的转运。我们已经通过shRNA方法获得了HO-2缺失的细胞系，或者通过CRISPR定向基因敲除(KO)获得了完全缺乏HO-2的细胞系，并证明逆转录病毒病毒粒子的产量急剧增加。我们现在将通过免疫荧光的固定切片检查和活细胞成像来表征有和没有HO-2的肉豆蔻酰化小鼠白血病病毒(MLV)Gag蛋白的细胞内转运。我们将通过对提取物进行沉降分析，然后用抗GAG抗体进行Western blotting来研究含有GAG的细胞内复合体的大小。我们将通过免疫共沉淀实验跟踪GAG多聚体的过程。我们将寻找与其已知的贩运伙伴(包括ABCE1、DDX6和MOV10)以及已知在病毒粒子组装和释放过程中起重要作用的蛋白质(特别是ESCRT和CHMP蛋白质)之间的GAG关联的变化。我们将通过RNA免疫沉淀(RIP)试验来评估GAG与病毒RNA的相互作用过程。这些实验应该让我们对HO-2在控制病毒颗粒组装中的GAG定位和功能方面的作用有一个全面的了解。这项工作将为针对逆转录病毒和其他编码肉豆蔻酰化蛋白的病毒的抗病毒治疗确定一个新的潜在靶点。此外，我们还将探讨HO-2在特定细胞肉豆蔻酰化蛋白运输中的作用。我们已经开发了一系列与HO-2结合的宿主蛋白，并将重点放在其中的两种蛋白上：Toll样受体适配器分子2(TRAM)，参与天然免疫信号；;和网状2，细胞内运输和促进膜弯曲的关键蛋白。我们将研究HO-2在控制TRAM的定位中的作用，TRAM与TLR4受体的联系，以及TRAM对内毒素(LPS)的反应。类似地，我们将测试HO-2在网状2定位于内质网中的作用。由于其在膜弯曲中的潜在作用，我们将测试网状2 KO对内质网结构和病毒粒子萌发的影响。这些实验将揭示HO-2在调节那些介导病毒限制和影响病毒粒子组装的特定宿主功能方面的新功能。这些发现具有很大的潜力，可以揭示调控或抑制病毒复制的全新方法。

We will explore the role of a novel host factor in retrovirus replication, heme oxygenase 2 (HO-2), which we discovered binds N-terminally myristoyled proteins and regulates their trafficking to the plasma membrane. We have generated cell lines deficient in HO-2 by shRNA methods, or entirely lacking HO-2 by CRISPR-directed knockout (KO), and documented dramatic increases in retrovirus virion production. We will now characterize the intracellular transport of the myristoylated murine leukemia virus (MLV) Gag protein with and without HO-2 by examination of fixed sections by immunofluorescence, and by live cell imaging. We will study the size of intracellular complexes containing Gag by sedimentation analysis of extracts, followed by Western blots probed with anti-Gag antibodies. We will follow the course of Gag multimerization by co-immunoprecipitation experiments. We will look for alterations in Gag association with its known trafficking partners (including ABCE1, DDX6, and MOV10), and with proteins known to be important in the process of virion assembly and release (notably the ESCRT and CHMP proteins). We will assess the course of Gag interaction with viral RNA by RNA-immunoprecipitation (RIP) assays. These experiments should give us a comprehensive view of the role of HO-2 in controlling Gag localization and function in virion assembly. The work will define a new potential target for antiviral therapies against retroviruses, and other viruses encoding myristoylated proteins. In addition, we will explore the role of HO-2 in trafficking of selected cellular myristoylated proteins. We have developed an extensive hit list of host proteins bound by HO-2 and will focus on two of these proteins: Toll-like receptor adaptor molecule 2 (TRAM), involved in innate immune signaling;; and reticulon 2, a key protein in intracellular trafficking and in promoting membrane curvature. We will characterize the role of HO-2 in controlling the localization of TRAM, the association of TRAM with the TLR4 receptor, and the signaling through TRAM in response to lipopolysaccharide (LPS). We will similarly test for the role of HO-2 in the localization of reticulon 2 to the endoplasmic reticulum. Because of its potential role in membrane bending, we will test for the effect of reticulon 2 KO on ER structure and virion budding. These experiments will reveal new functions of HO-2 in regulating those specific host functions that mediate virus restriction and impact virion assembly. The findings have great potential to reveal entirely new approaches to regulate or inhibit virus replication.