Mechanisms of viral protein trafficking in polarized insect cells

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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.