The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells. The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel, rather than in the gut. In this study, we used fall armyworm, Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis. We also assessed the rate of flux of the primary viral structural protein, VP4 fused to eGFP with a proline-rich linker (VP4-P-eGFP) through the gut epithelium ex vivo in an Ussing chamber. The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors. Bovine serum albumin (BSA) and eGFP were used as positive and negative control proteins, respectively. In contrast to BSA, which binds to multiple proteins on the brush border membrane, VP4-P-eGFP binding was specific to a protein of high molecular mass. Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP, with rapid transcytosis of VP4-P-eGFP within the first 30 min. In contrast to BSA which transcytosed following clathrin-mediated endocytosis, the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent. The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S. frugiperda.
A wide range of proteins move via vesicular transport across insect gut epithelial cells for release into the hemocoel. The utility of such transcytosed proteins for delivery of insectspecific neurotoxins from the gut into the hemocoel of aphids has recently been demonstrated for an aphid-transmitted, plant virus coat protein. Proteins that transcytose across the insect gut epithelium allow for appropriate delivery of toxins that are active within the hemocoel, providing a new approach toward development of pest resistant crops. We used an Ussing chamber to examine the transport efficiencies and mechanisms involved with the movement of a virus-derived coat protein and bovine serum albumin across the gut of Spodoptera frugiperda. Test proteins were labeled with fluorescein isothiocyanate or eGFP for ease of detection. There was wide variation in the efficacy of transport of the two proteins across the epithelial layer. The mechanisms involved with transport were investigated by addition of inhibitors into the Ussing chamber. While bovine serum albumen was taken up by clathrin-mediated endocytosis, uptake of the viral coat protein by the gut epithelial cell was dynamin dependent, but clathrin independent. Results are discussed in relation to the potential exploitation of these proteins and pathways for insect pest management.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.