Mechanisms of viral protein trafficking in polarized insect cells

节肢动物代表了地球上最大的动物生物量。它们的种群受到许多病毒性疾病的调节，它们携带许多导致植物和动物疾病的病毒。通常，病毒通过口服途径进入昆虫，并且必须穿透肠道细胞才能传播到其他组织。由于在这一领域知之甚少，这些研究将集中在病毒通过昆虫肠道细胞的具体机制。通常情况下，病毒会感染肠道细胞并在其中复制，新产生的颗粒会从另一侧发芽，进入昆虫的血液。这些研究将检查病毒及其组分如何以高度特异性的定向方式在细胞中移动，以便病毒可以进入昆虫的血液并最终感染其他组织。在这些研究的另一个组成部分中，将鉴定被细胞识别的病毒蛋白信号，用于招募到细胞运输途径中。这些研究利用了果蝇强大的遗传系统。该系统将与细胞培养系统和其他现代分子技术结合使用，以鉴定极性病毒通过昆虫肠道细胞运输所需的昆虫蛋白和病毒信号。这些研究将对各种病毒-昆虫相互作用产生影响，并应用于农业作物保护、林业、动物科学和人类健康等不同领域。此外，本科生和研究生的学生培训将支持本提案。病毒运输通过昆虫中肠上皮细胞是一个重要的感染步骤，大多数病毒感染昆虫，并为这些病毒的昆虫为媒介。然而，由于在昆虫肠道中进行实验操作的困难性，即使在最好的表征系统中，这个过程也没有得到很好的理解。这些研究结合了一个实验设计，利用两个强大的模型系统来识别参与病毒中肠运输的特定蛋白质，途径和信号。使用果蝇中肠细胞中病毒蛋白的异位表达，随后使用RNAi蝇株的综合文库进行RNAi筛选，将筛选候选途径以鉴定两种模型病毒蛋白（杆状病毒GP 64和VSV G）的极化运输所需的特定蛋白和途径。为了鉴定中肠运输所必需的病毒蛋白信号或基序，将在天然宿主昆虫鳞翅目粉纹夜蛾中分析杆状病毒GP 64蛋白。含有先前表征的包膜蛋白突变的病毒的现有文库将用于鉴定被识别用于招募至运输途径的病毒蛋白信号。病毒信号和宿主中肠蛋白以及极化运输途径的鉴定将为病原体-昆虫相互作用建立新的范例，并将极大地促进对病毒如何与作为宿主和载体的昆虫有效相互作用的理解。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Arthropods represent the largest animal biomass on earth. Their populations are regulated by many viral diseases, and they vector many viruses that cause diseases of plants and animals. Typically, a virus enters the insect by the oral route and must penetrate the cells of the gut to spread to other tissues. Because little is known in this area, these studies will focus on the specific mechanisms by which viruses move through the insect gut cells. Typically, a virus will infect and replicate in the gut cells and newly-produced particles will bud from the other side, into the insect's blood. These studies will examine how the virus and its components move through the cell in a highly specific directional manner, so that the virus can enter the insect's blood and eventually infect other tissues. In another component of these studies, viral protein signals that are recognized by the cell, for recruitment into cellular trafficking pathways will be identified. These studies capitalize on the powerful genetic system in the fruitfly, Drosophila melanogaster. This system will be used in combination with cell culture systems and other modern molecular techniques, to identify the insect proteins and viral signals that are required for polarized virus trafficking through insect gut cells. The studies will have impacts across a wide variety of virus-insect interactions, with applications in diverse areas such as agricultural crop protection, forestry, animal science, and human health. In addition, undergraduate and graduate student student training will be supported by this proposal.Viral transport across the insect midgut epithelium is an important infection step for most viruses that infect insects, and for those viruses vectored by insects. However, this process is not well-understood even in the best characterized systems due to the difficult nature of experimental manipulation in the insect gut. These studies incorporate an 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 comprehensive libraries of RNAi fly strains, candidate pathways will be screened to identify specific proteins and pathways necessary for polarized trafficking of two model viral proteins, baculovirus GP64 and VSV G. To identify viral protein signals or motifs necessary for midgut trafficking, the baculovirus GP64 protein will be analyzed in a natural host insect, the lepidopteran Trichoplusia ni. Existing libraries of viruses containing previously characterized envelope protein mutations will be used to identify the viral protein signals recognized for recruitment to trafficking pathways. The identification of viral signals and host midgut proteins and pathways for polarized trafficking will establish new paradigms for pathogen-insect interactions and will dramatically advance the understanding of how viruses productively interact with insects as hosts and vectors.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.