Fish TRIM35 negatively regulates the interferon signaling pathway in response to grouper nodavirus infection

Abstract Tripartite motif-containing protein 35 (TRIM35) has been demonstrated to exert critical roles in cancer, cell death and other multiple cell processes. However, the precisely roles of TRIM35 during virus infection still remained largely unknown. In the current study, we cloned a TRIM35 gene from orange spotted grouper (EcTRIM35) and uncovered its roles in response to nodavirus infection. EcTRIM35 encoded a 456-aa protein which showed 65% and 32% identity to large yellow croaker ( Larimichthys crocea ) and human ( Homo sapiens ), respectively. Structure prediction and amino acid alignment analysis indicated that EcTRIM35 contained three conserved domains, including RING domain, B-BOX and SPRY domain. In healthy grouper, the high expression level of EcTRIM35 could be detected in liver, spleen and intestine. After infection with red-spotted grouper nervous necrosis (RGNNV) and Singapore grouper iridovirus (SGIV) in GS cells, the transcript of EcTRIM35 was significantly up-regulated with the infection time increased. Under fluorescence microscopy, the bright fluorescence aggregates were observed in EcTRIM35 transfected cells, but the fluorescence distribution was obviously altered in the EcTRIM35-ΔRING transfected cells. After incubation with RGNNV, the overexpression of EcTRIM35 in vitro significantly enhanced the viral replication, evidenced by the enhancement of cytopathic effect (CPE) severity and the up-regulation of the viral gene transcription. Moreover, the ectopic expression of EcTRIM35 significantly decreased the expression of interferon signaling molecules or effectors. Further studies elucidated that EcTRIM35 overexpression significantly weakened the MAVS-, MITA- or TBK1-induced interferon immune response, but showed no effects on MDA5-induced immune response. Thus, our results will shed new lights on the roles of fish TRIM35 in innate immune response against grouper virus infection.