Abstract

Porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus that causes significant economic losses in the swine industry. It continues to pose a substantial threat to global swine production due to the limited efficacy of existing vaccines. Host-directed antiviral strategies targeting cellular pathways represent a promising approach to combat evolving viral strains. On the basis of our previous findings of a strong correlation between cholesterol transport and PEDV entry, we screened a panel of cholesterol-modulating compounds for anti-PEDV activity. We identified U18666A, an inhibitor of the Niemann-Pick C1 (NPC1) cholesterol transporter, as a potent and broad-spectrum inhibitor against multiple PEDV genotypes in both cell lines and porcine intestinal organoids. To validate NPC1 as a relevant pharmacological target, we utilized CRISPR/Cas9 to knock out NPC1 in Huh7 cells, which significantly inhibited PEDV infection. Conversely, forced expression of NPC1 in knockout cells partially restored viral infectivity. Using both authentic PEDV and PEDV pseudoviruses, we demonstrated that NPC1 facilitates the internalization of PEDV entry. Furthermore, co-immunoprecipitation and structural modeling revealed a direct interaction between NPC1 and the membrane-fusion subunit (S2) of the PEDV spike protein, as well as a role for NPC2 in this process, indicating that the NPC1/NPC2 cholesterol transport complex participates directly in viral entry. Additionally, exogenous cholesterol supplementation partially mitigated the inhibitory effect of U18666A, underscoring that functional intracellular cholesterol transport, not merely the presence of cholesterol, is required for efficient PEDV entry.