Usp3 negatively regulates Mda5-mediated antiviral response by attenuating its K63-linked ubiquitination in black carp

Melanoma differentiation-associated gene 5 (MDA5) is a critical cytosolic RNA sensor that initiates type I interferon (IFN) signaling upon viral infection, and its activity must be tightly regulated to maintain immune homeostasis. Ubiquitin-specific protease 3 (USP3) has been identified as a negative regulator of RLR-mediated IFN signaling in mammals, yet its role in teleost antiviral immunity remains unknown. In this study, we cloned and characterized usp3 (bcusp3) from black carp (Mylopharyngodon piceus). The full-length coding sequence of bcusp3 encodes a protein of 524 amino acids containing conserved ZnF-UBP and UCH domains. Overexpression of bcUsp3 significantly suppressed bcMda5-induced ifn promoter activation in a dose-dependent manner, whereas knockdown of bcUsp3 in MPF cells markedly enhanced ifn and interferon-stimulated gene (isg) expression and inhibited SVCV replication. Mechanistically, co-immunoprecipitation (Co-IP) assays revealed that bcUsp3 directly interacts with bcMda5. Furthermore, bcUsp3 over-expression reduced both total and K63-linked ubiquitination of bcMda5, indicating that bcUsp3 attenuates the K63-linked ubiquitination of bcMda5. Collectively, our findings demonstrate that bcUsp3 functions as a negative regulator of bcMda5-mediated Ifn signaling by attenuating its K63-linked ubiquitination, providing the first evidence for the regulatory role of Usp3 in teleost antiviral innate immunity.