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

我们将探讨一种新的宿主因子在逆转录病毒复制中的作用，血红素加氧酶2（HO-2），我们发现它结合N-末端肉豆蔻酰化蛋白并调节其向质膜的运输。我们已经通过shRNA方法产生了HO-2缺陷的细胞系，或者通过CRISPR定向敲除（KO）完全缺乏HO-2，并记录了逆转录病毒病毒粒子产量的显着增加。现在，我们将通过免疫荧光和活细胞成像检查固定切片来表征肉豆蔻酰化鼠白血病病毒（MLV）Gag蛋白（含和不含HO-2）的细胞内转运。我们将通过提取物的沉降分析研究含有Gag的细胞内复合物的大小，然后用抗Gag抗体探测蛋白质印迹。我们将通过免疫共沉淀实验跟踪Gag多聚化的过程。我们将寻找Gag与其已知的贩运伙伴（包括ABCE 1，DDX 6和MOV 10）以及已知在病毒体组装和释放过程中重要的蛋白质（特别是ESCRT和CHMP蛋白）的改变。我们将通过RNA免疫沉淀（RIP）试验评估Gag与病毒RNA相互作用的过程。这些实验应该给我们一个全面的看法HO-2的作用，在控制Gag的定位和功能的病毒体组装。这项工作将为抗逆转录病毒和其他编码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，我们将探讨HO-2在选定的细胞豆蔻酰化蛋白的运输中的作用。我们已经开发了一个广泛的命中清单的主机蛋白结合HO-2，并将集中在这些蛋白质中的两个：Toll样受体适配器分子2（TRAM），参与先天免疫信号传导; natural和reticulon 2，在细胞内运输和促进膜曲率的关键蛋白。我们将描述HO-2在控制TRAM定位中的作用，TRAM与TLR 4受体的关联，以及通过TRAM响应脂多糖（LPS）的信号传导。我们将同样测试HO-2在内质网2的定位中的作用。由于其在膜弯曲中的潜在作用，我们将测试reticulon 2 KO对ER结构和病毒体出芽的影响。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体组装的特异性宿主功能中的新功能。这些发现有很大的潜力揭示全新的方法来调节或抑制病毒复制。

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.