Cyclo-C stabilizes PEX13 to inhibit porcine epidemic diarrhea virus replication by blocking pexophagy-mediated disruption of antiviral innate immunity.

UNLABELLED: The persistent threat of porcine epidemic diarrhea virus (PEDV) to the global swine industry is compounded by high neonatal piglet mortality and the absence of effective antiviral therapies. Host-directed strategies that reinforce immunity offer a promising avenue to counter viral immune evasion. Through screening of an FDA-approved compound library, we identify the small-molecule cyclocytidine hydrochloride (Cyclo-C) as a potent inhibitor of PEDV replication that acts by stabilizing the peroxisomal biogenesis factor PEX13, a previously unrecognized host restriction factor. The antiviral activity of Cyclo-C is strictly PEX13-dependent, as it is completely abrogated in PEX13 knockout cells. Mechanistically, Cyclo-C disrupts the interaction between PEX13 and the viral nonstructural protein 8 (NSP8), thereby preventing NSP8-mediated PEX13 degradation and the subsequent induction of PI3K/AKT/mTOR-driven pexophagy. Preservation of peroxisomal integrity stabilizes the peroxisome-localized pool of MAVS, leading to a robust enhancement of type III interferon (IFN-III) responses that suppress viral replication. Critically, this mechanism translates in vivo, where Cyclo-C treatment of PEDV-challenged piglets significantly reduces mortality, lowers viral loads, and protects intestinal villus architecture. Our findings establish Cyclo-C as a first-in-class host-directed therapeutic candidate and validate the concept that pharmacological preservation of peroxisome-mediated innate immunity represents an effective antiviral strategy against enteric coronaviruses.

IMPORTANCE: The high genetic variability of porcine epidemic diarrhea virus (PEDV) limits current vaccine efficacy, and no antiviral therapeutics exist. Host-directed therapies targeting cellular pathways that viruses exploit for immune evasion offer an alternative approach. Here, we identify the FDA-approved compound Cyclo-C as a potent inhibitor of PEDV replication. Cyclo-C acts by stabilizing PEX13, a host protein that the virus degrades to evade immunity. By blocking viral protein NSP8 from binding PEX13, Cyclo-C prevents virus-induced pexophagy, thereby preserving peroxisomal integrity. This preserves peroxisome-localized MAVS and enhances type III interferon responses. In infected neonatal piglets, Cyclo-C reduced mortality and viral loads while protecting intestinal integrity. This study provides proof of concept that targeting peroxisomal immune regulation is a viable antiviral strategy and identifies Cyclo-C as a promising candidate for treating PEDV infection.