Bioinformatic analysis and functional characterization of the NPC2 gene in Apis mellifera during IAPV infection

This study aimed to investigate the molecular characteristics of NPC intracellular cholesterol transporter 2 (AmNPC2) in Apis mellifera and its potential regulatory role during Israeli acute paralysis virus (IAPV) infection. Through integrated approaches including bioinformatics analysis, quantitative real-time PCR (qRT-PCR), and RNA interference (RNAi), we examined the molecular features, tissue expression profile, and regulatory function of AmNPC2 in IAPV replication. Bioinformatics analysis revealed that the AmNPC2 gene contains a 465bp coding sequence (CDS) encoding a 154-amino acid protein with a predicted molecular weight of 16.8 kDa. Structural analysis revealed 14 phosphorylation sites, a transmembrane domain, and an N-terminal signal peptide (cleavage site between residues 17-18), indicating extracellular localization. Phylogenetic analysis showed that AmNPC2 clusters with NPC2 from Apis cerana, and secondary structure prediction identified 26 α-helices, 39 β-strands, 6 β-turns, and 83 random coils. RT-qPCR analysis demonstrated ubiquitous AmNPC2 expression across eight examined tissues in worker bees, with highest levels in the honey sac and trachea-tissues that also showed predominant IAPV tropism during natural infection. Experimental infection studies demonstrated that IAPV infection induces a significant upregulation of AmNPC2 gene expression in the host. RNAi-mediated knockdown of AmNPC2 led to more than 6-fold reduction in viral loads, suggesting that AmNPC2 facilitates IAPV infection, potentially through modulation of host lipid metabolism. Our results demonstrated that AmNPC2 may participate in IAPV infection by regulating host lipid metabolism. This study provided the first evidence for the potential role of NPC2 genes in honeybee viral pathogenesis, offering new theoretical insights into bee-virus interaction mechanisms.