Virus trafficking in insect midgut cells

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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.