The TLR9/NF-κB and cGAS/STING pathways mediate​ the assembly of the NLRP3 inflammasome induced by Singapore grouper iridovirus DNA.

Groupers are economically significant fish in southern China and Southeast Asia, where viral diseases severely hinder the sustainable development of aquaculture. Singapore grouper iridovirus (SGIV) is a highly pathogenic virus in groupers that can cause mass mortality upon infection. NOD-like receptor thermal protein domain associated protein 3 (NLRP3), a crucial component of innate immunity, plays vital roles in various viral infections. However, whether SGIV infection induces NLRP3 inflammasome activation and the precise mechanisms involved remain to be elucidated. In this study, we investigated the mechanism using an in vitro SGIV infection model of GS cells. Our findings demonstrated that SGIV infection significantly upregulated the transcription of NLRP3 and IL-1β. Additionally, SGIV induced pronounced aggregation of NLRP3 and enhanced the interaction between NLRP3 and ASC. Further investigation revealed that SGIV infection markedly upregulated TLR9 transcription while promoting nuclear translocation of NF-κB and enhancing its promoter activity. Transfection with SGIV genomic DNA in vitro also significantly upregulated transcription of TLR9, NLRP3, and IL-1β, along with NF-κB promoter activity. When TLR9 expression was knocked down by siRNA, NF-κB promoter activity and transcription levels of NLRP3 and IL-1β were substantially reduced. Similarly, the NF-κB inhibitor JSH-23 significantly suppressed NLRP3 and IL-1β transcription. Moreover, SGIV DNA upregulated transcription of cGAS and STING, while STING overexpression enhanced Caspase-1 activity and promoted IL-1β activation. This study first elucidates that the genomic DNA of SGIV may upregulate the transcription of IL-1β through the TLR9/NF-κB and cGAS/STING pathways, thereby inducing the assembly of the NLRP3 inflammasome.