Fish TRAF6 shapes interferon antiviral response by targeting the background MAVS for protein degradation

ABSTRACT The interferon (IFN)-mediated antiviral immune response is tightly regulated to prevent insufficient or excessive immune activation. In mammals, the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) is generally considered a positive regulator of IFN signaling although its role in fish may differ. In this study, zebrafish TRAF6 showed opposing regulatory effects on different retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling components in overexpression assays, but knockdown and knockout experiments further supported its overall negative regulatory role in the fish IFN response. To investigate this inhibitory mechanism, we uncovered a previously unrecognized role of zebrafish TRAF6 in promoting the degradation of mitochondrial antiviral-signaling protein (MAVS) under basal conditions in an E3 ligase activity-dependent manner. Zebrafish TRAF6 bound to the N-terminal caspase recruitment domain (CARD) of MAVS in the cytoplasm and promoted lysosome-dependent MAVS degradation. Lys286 and Lys293 contributed to this degradation and were required for TRAF6-enhanced K48-linked polyubiquitination of MAVS. Simultaneous mutation of both residues increased basal MAVS protein levels and enhanced its ability to activate the IFN response. Our data also suggest that TRAF6 may promote MAVS degradation through a proteasome-dependent pathway involving the C-terminal region of MAVS. TRAF6 knockout (TRAF6-KO) zebrafish exhibited significantly enhanced resistance to spring viremia of carp virus (SVCV) infection. Basal MAVS protein levels were also significantly higher in TRAF6-KO tissues than in wild-type (WT) zebrafish. Collectively, our results reveal a critical role of zebrafish TRAF6 in regulating basal MAVS expression to fine-tune IFN-mediated antiviral innate immunity. IMPORTANCE Interferon (IFN)-mediated innate immunity is essential for protecting fish against viral infection, but excessive IFN activation can also be harmful to the host. Here, we identify zebrafish TRAF6 as a dual regulator of IFN-mediated antiviral signaling. In overexpression assays, TRAF6 enhanced RIG-I-mediated signaling and increased K63-linked ubiquitination of the RIG-I signaling complex while suppressing MAVS-mediated signaling by promoting MAVS degradation through distinct degradation pathways. In vivo , TRAF6 deficiency enhanced antiviral gene expression, reduced infectious viral titers, and significantly improved survival following SVCV infection. These findings reveal a previously unrecognized dual regulatory role for TRAF6 in fish antiviral immunity and highlight the importance of TRAF6-mediated MAVS degradation under basal conditions in regulating IFN-mediated antiviral responses.