Porcine IFITM1 Promotes PEDV Entry by Associating with Viral Structural Proteins and Modulating Cholesterol-Dependent Caveolae-Mediated Endocytosis

Porcine epidemic diarrhea virus (PEDV) severely threatens the global swine industry and causes massive economic losses due to high morbidity and mortality in neonatal piglets. To date, key host receptors and molecular determinants regulating PEDV entry remain poorly characterized. Here, we found that overexpression of porcine interferon-induced transmembrane protein 1 (pIFITM1) in LLC-PK1, IPEC-J2, and Huh7 cells enhanced PEDV-HM infection. Conversely, knockout of endogenous pIFITM1 suppressed PEDV-HM infection, and this phenotype was rescued by pIFITM1 reconstitution. Additionally, pIFITM1 interacted with the PEDV spike, envelope, and membrane proteins and promoted PEDV attachment and internalization. As PEDV invades host cells via caveolae-mediated endocytosis, we further confirmed that pIFITM1 interacted with caveolin-1 (CAV1) and modulated plasma membrane cholesterol distribution to promote PEDV entry through this pathway. Mechanistically, deletion of the conserved intracellular loop (CIL) abolished the proviral effect of pIFITM1. Notably, site-directed mutagenesis of lysine residues K62 and K83 within the CIL abolished its proviral function, suggesting that these two residues are essential for pIFITM1-mediated promotion of PEDV entry. In summary, our study demonstrates that pIFITM1 acts as a critical proviral factor that facilitates PEDV entry by binding viral structural proteins and regulating the cholesterol–CAV1 complex. This study also identifies pIFITM1 as a promising target for developing anti-PEDV interventions.