The cholesterol transporter Niemann-Pick C1 facilitates the entry of porcine epidemic diarrhea coronavirus.

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. Collectively, our findings elucidate a mechanism wherein PEDV co-opts the host NPC1/NPC2 cholesterol transport machinery for efficient internalization, thereby identifying this pathway as a promising target for the development of broad-spectrum host-directed antivirals. Porcine epidemic diarrhea virus (PEDV) causes severe enteric disease in swine and continues to impose substantial economic losses on the global pig industry. Understanding the host determinants that govern PEDV entry is therefore critical for elucidating the mechanisms of infection. Here, we show that efficient PEDV entry is closely associated with intact and functional intracellular cholesterol transport in host cells. We identified the cholesterol transporter Niemann-Pick C1 (NPC1) as a critical host factor required for PEDV entry and demonstrated that NPC1 facilitates viral internalization through direct interaction with the S2 subunit of the PEDV spike protein. In addition, coordinated functions of the NPC1/NPC2 cholesterol transport system are involved in this process. By revealing the functional dependence of PEDV entry on host cholesterol trafficking, this study highlights this pathway as a potential target for therapeutic intervention.