IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus's impact on the swine industry. IMPORTANCE: Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.