Ring finger protein 5 mediates STING degradation through ubiquitinating K135 and K155 in a teleost fish

Stimulator of interferon genes (STING) is a key connector protein in interferon (IFN) signaling, crucial for IFN induction during the activation of antiviral innate immunity. In mammals, ring finger protein 5 (RNF5) functions as an E3 ubiquitin ligase, mediating STING regulation through K150 ubiquitylation to prevent excessive IFN production. However, the mechanisms underlying RNF5's regulation of STING in teleost fish remain unknown. This study investigated the regulatory role of the mandarin fish ( ) RNF5 ( RNF5) in the STING-mediated antiviral immune response and identified the specific regulatory sites on STING. Furthermore, an examination of RNF5 expression patterns in virus-infected cells revealed its responsiveness to mandarin fish ranavirus (MRV) infection. The ectopic expression of RNF5 suppressed STING-mediated IFN signaling and facilitated MRV replication. Co-immunoprecipitation experiments indicated an interaction between RNF5 and STING. The further experiments demonstrated that RNF5 exerted its inhibitory effect by promoting the degradation of STING, which was observed to be blocked by MG132 treatment. Ubiquitination assays with various STING mutants showed that RNF5 catalyzed the ubiquitination of STING at K135 and K155 residues. Furthermore, we provided evidence that RNF5 significantly attenuated STING-dependent antiviral immunity by targeting negative regulators within the STING signaling cascade. This study underscored that RNF5 negatively regulated the STING-mediated IFN signaling pathway in mandarin fish, attenuated STING's antiviral activity, and facilitated STING degradation via the ubiquitin-proteasome pathway at two novel lysine sites (K135 and K155). Our work offered valuable insights into the regulatory mechanisms of STING-mediated signaling in teleost fish, paving the way for further research.