Screening and functional analysis of a host membrane protein interacting with the structural protein VP1 of deformed wing virus

Abstract

Introduction Deformed wing virus (DWV) is a major viral pathogen infecting Western honeybees (Apis mellifera). However, the interactions between viral structural proteins and host proteins during infection remain poorly understood. In this study, we used a yeast membrane protein two-hybrid system to screen for host membrane proteins that interact with the DWV structural protein VP1. The identified interactions were further functionally characterized using glutathione S-transferase (GST) pull-down, co-immunoprecipitation (Co-IP) and RNA interference (RNAi) assays. Methods The bait plasmid pBT3-STE-VP1, carrying the VP1 gene, was screened against a complementary DNA library of Western honeybee proteins. Among the 22 candidate host proteins identified, a neuropeptide capa (NCR) like G-protein-coupled receptor was selected for further analysis. The interaction between DWV VP1 and NCR-like was confirmed using GST pull-down assays, Co-IP and co-expression. Using healthy bee pupae as experimental subjects, RNAi mediated knockdown of the NCR-like gene was performed to investigate its effect on the expression of antimicrobial peptides (AMPs), including Defensin-1 and Hymenoptaecin. Furthermore, RNAi was used to silence the NCR-like gene in DWV-infected bee pupae to evaluate its impact on viral replication. Results Viral replication levels increased approximately sixfold compared with the infection-only control group. These results indicate that DWV VP1 interacts with the host NCR-like protein to regulate the expression of immune effectors, particularly AMPs, thereby influencing DWV replication. Discussion This study provides initial mechanistic insights into interactions between DWV and its host, as well as the viral pathogenic mechanisms.