Design and Validation of Plasmid Vectors for Characterizing Protein–Protein Interactions in Spodoptera frugiperda Insect Cells

Abstract

There are limited molecular biology resources for interrogating protein–protein interactions (PPIs) in insect cells. To address this deficiency, we developed plasmid vectors for localization, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation (co-IP) assays in Sf9 insect cells. Plasmids were designed to express a protein of interest as a fusion with epitope tags and autofluorescent proteins using the Gateway cloning system. Two robust interactors were utilized to validate this system: the nucleoprotein (N) and the phosphoprotein (P) of maize mosaic virus. The viral N was fused with the carboxy-terminal portion of eYFP and a FLAG epitope tag, and P was fused with the amino-terminal portion of eYFP and a myc epitope tag. The two expression plasmids were cotransfected into Sf9 cells, fluorescence microscopy was used to visualize BiFC, and co-IP was performed to confirm that this system was sensitive enough to detect PPI between the two proteins. BiFC was seen in cells cotransfected with N and P, and co-IP validated the interaction. This plasmid-based system can be used to investigate a variety of PPIs that occur in insects. We validated viral protein interactions that occur in the insect vector, which provides further insights into the biology of rhabdoviruses that are transmitted by insects. The ability to express viral and insect proteins in insect cells for studying PPIs with this streamlined system represents an advancement for protein research in insects. Future work will focus on identifying interacting viral and host proteins and discovery of targets for control of viruses and insect vectors. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .