Baculovirus envelope proteins and insect cells

描述（由申请人提供）：杆状病毒是一种大型DNA病毒，是昆虫的剧毒病原体，它们是研究病毒与宿主相互作用的重要模型。此外，杆状病毒AcMNPV已被开发为哺乳动物细胞表达的转导载体，在高通量筛选和人类基因治疗等领域具有重要应用。AcMNPV出芽病毒粒子（BV）通过主要的包膜糖蛋白GP64介导病毒进入。GP64的受体尚不清楚。GP64也是有效的病毒粒子出芽和子代病毒产生所必需的。在AcMNPV的自然宿主中，GP64蛋白在昆虫传播的初始阶段也起着关键作用。GP64靶向极化中肠上皮细胞的基膜，这种靶向似乎指导出芽和感染传播到昆虫血中。在拟建的研究中，我们的工作将集中在三个具体领域：1)病毒受体结合；2)昆虫极化中肠上皮细胞的包膜蛋白靶向研究；3)病毒粒子的组装和出芽。病毒受体结合的研究将集中在GP64受体结合域和宿主细胞受体的鉴定上。我们还将鉴定GP64中肠靶向信号和参与该过程的相互作用蛋白。由于GP64对于有效的病毒粒子出芽至关重要，我们将鉴定GP64出芽结构域以及在组装和出芽过程中与GP64相互作用的病毒和/或细胞蛋白。在这些研究中，我们将使用表达GP64的工程细胞系和我们最近开发的强大的遗传系统，用修饰形式的GP64取代病毒基因组中的野生型GP64。利用这些强大的遗传工具和一系列功能分析，我们将在出芽病毒和病毒感染周期的背景下检查GP64的功能。这些研究将解决重要的核心问题，促进我们对杆状病毒与宿主受体的相互作用，病毒通过昆虫极化中肠上皮细胞的运动以及病毒出芽机制的理解。此外，这些研究的许多结果将直接适用于杆状病毒在哺乳动物细胞中的新的和令人兴奋的生物技术应用。

DESCRIPTION (provided by applicant): Baculoviruses are large DNA viruses that are virulent pathogens of insects and they serve as important models for studies of virus-host interactions. In addition, the baculovirus AcMNPV has been developed as a transduction vector for mammalian cell expression, with important applications in areas such as high throughput screening and the potential for use in human gene therapy. Viral entry by AcMNPV budded virions (BV) is mediated by the major envelope glycoprotein, GP64. The receptor for GP64 is not known. GP64 is also necessary for efficient virion budding and progeny virus production. In natural hosts of AcMNPV, the GP64 protein also plays a critical role in the initial phase of transmission in the insect. GP64 is targeted to basal membranes in polarized midgut epithelial cells and this targeting appears to direct budding and transmission of infection into the insect hemocoel. In the proposed studies, our work will be concentrated in three specific areas: 1) Viral receptor binding; 2) Envelope protein targeting in insect polarized midgut epithelial cells; and 3) Virion assembly and budding. Studies of viral receptor binding will focus on identification of the GP64 receptor binding domain and the host cell receptor. We will also identify the GP64 midgut targeting signal and interacting proteins involved in this process. Because GP64 is critical for efficient virion budding, we will identify the GP64 budding domain as well as viral and/or cellular proteins that interact with GP64 during assembly and budding. For these studies, we will use an engineered cell line expressing GP64, and a powerful genetic system that we recently developed, to replace wild type gp64 in the viral genome with modified forms of gp64. Using these powerful genetic tools with a range of functional assays, we will examine GP64 function in the context of budded virions and the viral infection cycle. These studies will address important and central questions, advancing our understanding of baculovirus interactions with host receptors, virus movement through insect polarized midgut epithelial cells, and the mechanism of virus budding. In addition, results of many of these studies will be directly applicable to new and exciting biotechnological applications of baculoviruses in mammalian cells.