NNV mediates the activation of the NLRP3 inflammasome through mtDNA release and oxidative DNA damage.

The NLRP3 inflammasome is a cytoplasmic multiprotein complex that is activated under stress or infection conditions. It promotes the maturation of IL-1β by cleaving pro-Caspase-1, thereby playing a critical role in host innate immune responses. Various viruses can differentially activate NLRP3 through distinct mechanisms, including potassium ion efflux and reactive oxygen species (ROS) accumulation. Mitochondrial DNA (mtDNA) has been demonstrated to be involved in virus-induced inflammasome activation and subsequent inflammatory responses. Red-spotted grouper nervous necrosis virus (RGNNV) is a highly pathogenic virus affecting groupers, causing extremely high fish mortality and substantial economic losses. Nevertheless, the regulatory mechanisms underlying NLRP3 assembly during NNV infection remain to be elucidated. This study aimed to elucidate the molecular mechanisms by which NNV activates the NLRP3 inflammasome. Our results demonstrated that NNV infection significantly upregulated the expression of NLRP3 and IL-1β, promoted punctate aggregation of NLRP3, and enhanced the interaction between EcNLRP3 and EcASC in GS cells. QRT-PCR, Western blotting, and luciferase reporter assays confirmed that NNV infection activated the TLR9/NF-κB pathway. Further investigation showed that NNV infection induced the release of mtDNA from GS cells, and transfection of exogenous mtDNA significantly upregulated the transcription of TLR9, NLRP3, and IL-1β. Moreover, NNV infection also induced ROS production and oxidative DNA damage. Treatment with the mitochondria-targeted antioxidant Mito-tempo significantly attenuated NNV-induced mtDNA release and oxidative DNA damage, and inhibited caspase-1 activity as well as the protein expression of Pro-IL-1β and IL-1β. Collectively, these findings suggest that NNV infection may participate in NLRP3 inflammasome activation by inducing mtDNA release and oxidative DNA damage, providing new insights into the mechanisms of NNV-induced inflammation.