CYLD通过阻断病毒复制及病毒引发的神经细胞中GSDMD依赖性细胞焦亡，抑制日本脑炎病毒的致病进程。

Abstract

JEV downregulates CYLD. • CYLD inhibits JEV replication (IFN-independent) and GSDMD pyroptosis. • CYLD deubiquitinase activity is essential for anti-JEV protection. • Spermidine-induced CYLD upregulation alleviates JEV pathogenesis via JNK/p38/NLRP3 inhibition. 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 illu