DAPK3 activates STING to enhance antiviral response via IRF3 and NF-κB pathways in teleosts

Stimulator of interferon genes (STING) is critical for the induction of type I interferons (IFN-I) and inflammatory responses against viral infection. The activity of STING is tightly regulated by various post-translational modifications (PTMs) to effectively defend against viral infection or prevent excessive immune responses. However, the regulatory mechanisms of STING activity remain largely unknown in teleosts. In this study, we discovered that zebrafish death-associated protein kinase 3 (DAPK3) enhances the STING-mediated antiviral innate immune response through two synergistic mechanisms: the induction of an interferon regulatory factor 3 (IRF3)-dependent IFN-I response and a nuclear factor kappa B (NF-κB)-dependent pro-inflammatory cytokine response. Specifically, viral stimulation triggers the expression of DAPK3, resulting in its autophosphorylation at Ser21 and Thr87. Activated DAPK3 subsequently interacts with STING, promoting its phosphorylation. Moreover, DAPK3 also recruits tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase, thereby increasing K63-linked polyubiquitination of STING. Subsequently, activated STING orchestrates two key responses: (i) it recruits TBK1 to enhance IRF3 phosphorylation and dimerization, thereby promoting IFN-I production; (ii) it recruits the IKK complex (IKKα/IKKβ) to activate p65-mediated expression of pro-inflammatory cytokines (IL-6, IL-1β, TNFα), ultimately inducing apoptosis. At the cellular level, DAPK3 overexpression significantly enhances cellular antiviral capacity and inhibits viral proliferation. In vivo, zebrafish embryos injected with DAPK3 mRNA display reduced lethality and decreased viral mRNA levels compared to controls. This is accompanied by the induction of immune-related genes and pro-inflammatory cytokines. Conversely, knocking down DAPK3 in zebrafish enhances spring viremia of carp virus (SVCV)-induced lethality and viral proliferation, a phenomenon associated with a reduction in immune-related genes and pro-inflammatory cytokines. Collectively, these findings underscore the critical role of zebrafish DAPK3 in enhancing the antiviral response through two synergistic mechanisms that target STING.