Grouper ubiquitin-specific protease 14 promotes iridovirus replication through negatively regulating interferon response

Abstract

Ubiquitin-specific protease 14 (USP14), one of the USP family members which belong to deubiquitinating enzymes (DUBs), plays a key role in maintaining cellular protein homeostasis by trimming ubiquitin chains from their substrates. However, the roles of USP14 in response to virus infection still remains largely unknown. In the current study, a USP14 homolog from orange spotted grouper (EcUSP14) was cloned and its roles in innate immune response were investigated. EcUSP14 was composed of 1479 base pairs encoding a 492-amino acid (aa) polypeptide. Sequence analysis indicated that EcUSP14 shared 96.14% and 81.30% identity to USP14 of bicolor damselfish (Stegastes partitus) and humans (homo sapiens), respectively. EcUSP14 contains conserved ubiquitin-like (UBL) domain (aa 3–76) and peptidase-C19A domain (aa 106–481). In response to Singapore grouper iridovirus (SGIV) infection in vitro, EcUSP14 was significantly up-regulated. Subcellular localization showed that EcUSP14 was predominantly localized in the cytoplasm of grouper spleen (GS) cells and mostly co-localized with the viral assembly sites after SGIV infection. The ectopic expression of EcUSP14 significantly promoted the replication of SGIV, as demonstrated by the accelerated progression of severity of cytopathic effect (CPE), the increased viral gene transcription and viral protein synthesis during infection. Consistently, treatment with IU1, a USP14 specific inhibitor, significantly inhibited the replication of SGIV, suggesting that USP14 function as a pro-viral factor during SGIV replication. Further analysis showed that EcUSP14 overexpression decreased the promoter activities of interferon (IFN)-1, IFN-3, IFN-stimulated response element (ISRE), and nuclear factor of kappa B (NF-κB). Furthermore, the ectopic expression of EcUSP14 decreased the activities of IFN-1 promoter evoked by TANK-binding kinase (TBK)-1 and melanoma differentiation-associated protein (MDA)-5, but not stimulator of interferon genes (STING). Thus, we speculated that EcUSP14 facilitated virus replication by negatively regulating the IFN response. Taken together, our results firstly demonstrated that fish USP14 functioned as a pro-viral factor by negatively regulating interferon response against virus infection.