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

ABSTRACT 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.