Shotgun Proteomic Identification of Amphotericin B Targets in Late Endosomes of Influenza A Virus-Infected A549 Cells

Amphotericin B (AmB), a polyene macrolide antifungal, is well known for its ability to bind ergosterol and cholesterol; however, its interactions with host proteins remain poorly characterized. In this study, we aimed to identify AmB-binding targets in A549 cells infected with the influenza A virus (H1N1) strain A/PR/8/34 (PR8). We isolated late endosomal fractions and performed a biotin–streptavidin pulldown using a biotinylated AmB probe, with specificity confirmed by competition with excess free AmB. The proteins enriched from these fractions were then analyzed by shotgun LC–MS/MS, revealing a set of late endosomal proteins that specifically interact with AmB during influenza A virus infection.