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.

泛素特异性蛋白酶14（USP14），属于去泛素化酶（DUBs）的USP家族成员之一，通过从其底物修剪泛素链来维持细胞蛋白质稳态中起着关键作用。但是，USP14在响应病毒感染中的作用仍然很大程度上未知。在当前的研究中，从橙色斑斑石斑鱼（EcUSP14）克隆了USP14同源物，并研究了其在先天免疫应答中的作用。EcUSP14由编码492个氨基酸（aa）多肽的1479个碱基对组成。序列分析表明，EcUSP14与USP14的双色雀鲷（Stegastes partitus）和人（智人）具有96.14％和81.30％的同一性）， 分别。EcUSP14包含保守的泛素样（UBL）域（aa 3–76）和肽酶-C19A域（aa 106–481）。响应新加坡石斑鱼虹膜病毒（SGIV）的体外感染，EcUSP14明显上调。亚细胞定位表明EcUSP14主要位于石斑鱼脾（GS）细胞的细胞质中，并在SGIV感染后与病毒装配位点共定位。EcUSP14的异位表达可显着促进SGIV的复制，这可通过细胞病变效应（CPE）严重程度的加速发展，感染过程中病毒基因转录的增加和病毒蛋白的合成来证明。一致地，用USP14特异性抑制剂IU1进行的治疗显着抑制了SGIV的复制，这表明USP14在SGIV复制过程中起了促病毒因子的作用。进一步的分析表明，EcUSP14过表达会降低干扰素（IFN）-1，IFN-3，IFN刺激的反应元件（ISRE）的启动子活性，κB）。此外，EcUSP14的异位表达降低了TANK结合激酶（TBK）-1和黑色素瘤分化相关蛋白（MDA）-5引起的IFN-1启动子的活性，但没有刺激干扰素基因（STING）。因此，我们推测EcUSP14通过负调节IFN反应来促进病毒复制。两者合计，我们的结果首先证明鱼USP14通过负调节干扰素对病毒感染的应答而起到促病毒作用。