CYLD restricts Japanese encephalitis virus pathogenic progression via blocking viral replication and virus-triggered...

Japanese encephalitis virus (JEV), a typical mosquito-borne neurotropic pathogen, triggers neuronal cell death and robust inflammatory responses within the central nervous system, posing severe threats to public health and animal husbandry worldwide. The host protein cylindromatosis (CYLD) acts as a vital deubiquitinating enzyme and negatively modulates a spectrum of inflammatory signaling cascades via its deubiquitinase activity; nevertheless, its functional role and underlying mechanism during JEV infection remain poorly defined. The present study was designed to explore the regulatory effects and molecular mechanism of CYLD in JEV replication as well as virus-elicited neuronal pyroptosis. Our results revealed that JEV infection markedly downregulated endogenous CYLD expression in a time-dependent fashion. Functional validation experiments demonstrated that CYLD overexpression remarkably restrained JEV replication, while CYLD knockdown exerted the opposite pro-viral effect, and such regulatory functions were independent of the interferon signaling pathway. Additionally, ectopic CYLD expression effectively mitigated JEV-triggered neuronal pyroptosis, whereas CYLD deficiency significantly exacerbated this pyroptotic process. Notably, the inhibitory effects of CYLD on viral replication and neuronal pyroptosis were completely abrogated by the application of deubiquitinase activity-deficient mutants and truncated CYLD constructs, confirming that the biological functions of CYLD are strictly dependent on its deubiquitinase active domain. Consistently, in vivo mouse infection models further validated the critical involvement of CYLD in JEV pathogenicity. Mechanistically, combined in vivo and in vitro assays illustrated that CYLD alleviates JEV-induced neuronal pyroptosis through targeted inhibition of the JNK/p38 MAPK signaling pathway and subsequent suppression of downstream NLRP3 inflammasome activation. Collectively, this study for the first time identifies CYLD as a pivotal negative regulator of neuronal pyroptosis upon JEV infection. CYLD exerts prominent neuroprotective effects and restricts JEV replication via suppressing NLRP3 inflammasome-dependent pyroptosis, which sheds new light on the molecular pathogenesis of Japanese encephalitis and provides a promising novel therapeutic target for the prevention and clinical treatment of JEV encephalitis.