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

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.