Negative regulation of the interferon response by finTRIM82 in the orange spotted grouper.

Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune response against grouper virus infection.