Virus trafficking in insect midgut cells

节肢动物是地球上生物量最大的动物。它们的数量受许多病毒性疾病的控制，节肢动物也传播许多引起动植物重要疾病的病毒。因此，自然界中节肢动物与病毒之间的相互作用是普遍存在的，也是重要的。在大多数昆虫-病毒相互作用中，病毒通过口腔途径进入昆虫，必须穿透昆虫肠道的细胞，才能进入昆虫的其他细胞和组织。虽然这是生物相互作用的关键步骤，但人们对病毒如何在昆虫中肠细胞中移动知之甚少。拟议的研究将检查这些相互作用，重点关注定向（极化）病毒通过昆虫中肠上皮细胞的转运机制。对于这些研究，将使用培养细胞和转基因果蝇的结合，并结合各种遗传和分子方法，发现与昆虫肠道细胞相互作用并指导病毒蛋白的细胞蛋白。这些关于昆虫中肠中病毒-昆虫相互作用的研究的广泛影响将包括在农业作物保护、林业、动物科学和人类医学等不同领域的应用。了解特定的病毒信号和相互作用的中肠细胞蛋白以及调节或限制病毒通过中肠细胞运输的途径，可用于增强有益病毒（如农业中的生物防治剂）的感染，并抑制植物和动物疾病的昆虫媒介中的病毒传播。许多动植物病毒是由昆虫传播的，昆虫也被一些致病病毒感染。通常，在病毒被口服后，病毒进入并在昆虫中肠极化的上皮细胞中复制。病毒成分的极化运输将病毒颗粒或组件运送到中肠细胞的基膜，在那里病毒可以组装并通过出芽离开细胞。即使在最具特征的系统中，病毒在昆虫中肠上皮细胞中的转运也不是很清楚，而且由于在该组织中进行实验操作的困难，对昆虫肠道中极化蛋白转运的机制性质知之甚少。该项目将利用创新的实验设计，利用两个强大的模型系统来识别涉及病毒-中肠运输的特定蛋白质、途径和信号。利用病毒蛋白在果蝇中肠细胞中的异位表达，然后使用RNAi蝇株文库进行RNAi筛选，并对果蝇细胞系进行高通量分析，筛选整个候选途径，以确定两种模型病毒蛋白在中肠中极化运输所需的特定蛋白质和途径。为了确定中肠运输所需的蛋白质信号，将分析一种被广泛研究的病毒蛋白（杆状病毒GP64）在一种天然寄主昆虫，鳞翅目毛虫中。鉴定介导和调节靶向运输的病毒蛋白信号、中肠蛋白和途径将为理解病虫相互作用提供新的范例。

Arthropods represent the largest animal biomass on earth. Their populations are regulated by many viral diseases, and arthropods also vector many viruses that cause important diseases of plants and animals. Thus, interactions between arthropods and viruses in nature are ubiquitous and important. In most insect-virus interactions, a virus enters the insect by the oral route and must penetrate the cells of the insect's gut in order to have access to the other cells and tissues of the insect. Although it is a critical step in the biological interaction, very little is known about how viruses move through insect midgut cells. The proposed studies will examine those interactions, focusing on the mechanisms of directional (polarized) viral transit through the epithelial cells of the insect midgut. For these studies, a combination of cultured cells and transgenic Drosophila will be used, and combined with a variety of genetic and molecular approaches, to discover the cellular proteins that interact with and direct viral proteins across the insect gut cells. Broader impacts of these studies of virus-insect interactions in the insect midgut will include applications in diverse areas such as agricultural crop protection, forestry, animal science, and human medicine. Understanding the specific viral signals and interacting midgut cell proteins and pathways that regulate or restrict virus trafficking through midgut cells can be used to both enhance infection by beneficial viruses (such as biocontrol agents in agriculture) and to inhibit viral transmission in insect vectors of plant and animal diseases. Many plant and animal viruses are vectored by insects, and insects are also infected by a number of pathogenic viruses. Typically, after the virus is consumed orally, the virus enters and replicates in polarized epithelial cells of the insect midgut. Polarized trafficking of viral components delivers the virus particle or components for assembly, to the basal membranes of the midgut cell where the virus may assemble and exit the cell by budding. Virus transport across insect midgut epithelial cells is not well understood even in the best characterized systems, and little is known about the mechanistic nature of polarized protein transport in the insect gut, due to the difficult nature of experimental manipulation in this tissue. This project will utilize an innovative experimental design that leverages two powerful model systems for identifying specific proteins, pathways, and signals involved in virus-midgut trafficking. Using ectopic expression of viral proteins in Drosophila midgut cells followed by RNAi screens using libraries of RNAi fly strains, and high throughput analysis of a Drosophila cell line, entire candidate pathways will be screened to identify specific proteins and pathways necessary for polarized trafficking of two model viral proteins in the midgut. To identify protein signals necessary for midgut trafficking, an extensively studied viral protein (baculovirus GP64) will be analyzed in a natural host insect, the lepidopteran Trichoplusia ni. The identification of viral protein signals and midgut proteins and pathways that mediate and regulate targeted trafficking will generate new paradigms for understanding pathogen-insect interactions.