Grouper OTUB1 and OTUB2 promote red-spotted grouper nervous necrosis virus (RGNNV) replication by inhibiting the host innate immune response.

Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro, and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques.

Fish RIP1 Mediates Innate Antiviral Immune Responses Induced by SGIV and RGNNV Infection.

Receptor interacting protein 1 (RIP1) is an essential sensor of cellular stress, which may respond to apoptosis or cell survival and participate in antiviral pathways. To investigate the roles of fish RIP1 in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, a RIP1 homolog from orange-spotted grouper (Epinephelus coioides) (EcRIP1) was cloned and characterized. EcRIP1 encoded a 679 amino acid protein that shares 83.28% identity with that of Perca flavescens and contained a homologous N-terminal kinase (S-TKc) domain, a RIP isotype interaction motif (RHIM), and a C-terminal domain (DD). EcRIP1 was predominantly detected in immune tissues, and its expression was induced by RGNNV or SGIV infection in vitro. Subcellular localization showed that EcRIP1 was distributed in the cytoplasm with point-like uniform and dot-like aggregation forms. Overexpression of EcRIP1 inhibited SGIV and RGNNV replication and positively regulated the expression levels of interferon (IFN) and IFN-stimulated genes and pro-inflammatory factors. EcRIP1 may interact with grouper tumor necrosis factor receptor type 1-associated DEATH domain protein (EcTRADD) to promote SGIV-induced apoptosis, and interact with grouper Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (EcTRIF) and participate in Myeloid Differentiation Factor 88 (MyD88)-independent toll-like receptor (TLR) signaling. EcRIP1 may also interact with grouper tumor necrosis factor receptor-associated factors (TRAFs) as intracellular linker proteins and mediate the signaling of various downstream signaling pathways, including NF-κB and IFN. These results suggest that EcRIP1 may inhibit SGIV and RGNNV infection by regulating apoptosis and various signaling molecules. Our study offers new insights into the regulatory mechanism of RIP1-related signaling, and provides a novel perspective on fish diseases mediated by RIP1.

Characterization of orange-spotted grouper (Epinephelus coioides) ASC and caspase-1 involved in extracellular ATP-mediated immune signaling in fish.

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. Caspase-1 is a member of inflammatory caspases that play important roles in the innate immune system. However, few studies have been performed in lower vertebrates such as teleosts and implications of extracellular ATP-mediated immune signalling in fish. Here we identified and characterized novel ASC and caspase-1 genes (namely EcASC and EcCaspase-1) from the orange-spotted grouper (Epinephelus coioides). EcASC and EcCaspase-1 encode 204- and 388-aa proteins which shared 55.34% and 72.89% identity with those in Siniperca chuatsi and Perca flavescens, respectively. EcASC contained a PYRIN domain (aa 5-82) and CARD domain (aa 107-201). EcCaspase1 contained a CARD domain (aa 1-88) and a CASc domain (aa 127-376). Both EcASC and EcCaspase-1 were distributed in all tissues tested in the healthy grouper. The expression of EcASC and EcCaspase-1 was significantly upregulated in response to ATP infection. Subcellular localization analysis showed that EcCaspase-1 exhibited a clear distribution in both cytoplasm and nucleus. In contrast, EcASC was observed in the cytoplasm as speck-like structures, which are called "pyroptosomes". EcCaspase-1 co-localized with the spot-like protein (EcASC). Overexpression of EcASC and EcCaspase-1 inhibited NF-κB activation and promoted P53 activation in grouper spleen (GS) cells. Extracellular ATP was an effective signaling molecule that activates the innate immune response, rapidly upregulating the expression of EcASC and EcCaspase1, and enhancing their promotion of proinflammatory cytokine expression in GS cells. Both EcASC and EcCaspase-1 promoted ATP-induced apoptosis. Our results suggested that the interactions of inflammatory EcCaspase-1 with EcASC proteins were associated with extracellular ATP-mediated immune signaling in fish.

Grouper TRADD Mediates Innate Antiviral Immune Responses and Apoptosis Induced by Singapore Grouper Iridovirus (SGIV) Infection.

Tumor necrosis factor (TNF) receptor type 1-associated DEATH domain protein (TRADD) is a TNFR1-associated signal transducer and an essential component of the TNFR1 complex that is involved in activating both apoptotic and nuclear factor (NF)-κB pathways as an adaptor. It also is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with virus as an essential component of the antiviral response. To date, few studies have investigated the function of TRADD in lower vertebrates and its antiviral response to DNA virus infection. In the present study, a TRADD gene (named as EcTRADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The full-length cDNA of EcTRADD consists of 1,370 base pairs (bp) and contains a 44 bp 5'-terminal untranslated region (UTR), a 450 bp 3'-UTR including a poly (A) tail, and an 876 bp open reading frame encoding a putative 291 amino acid protein. EcTRADD has two conserved domains of N-terminal domain (TRADD-N) and a death domain (DD). EcTRADD was detected in all examined tissues. EcTRADD was up-regulated in the spleen after infection with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that EcTRADD and EcTRADD-DD exhibited a clear pattern of discrete and interconnecting cytoplasmic filaments resembling the death-effector filaments, while EcTRADD-N was observed in the cytoplasm. After infection with SGIV, EcTRADD, and EcTRADD-DD were transferred to the nucleus. Overexpression of EcTRADD and its domains inhibited replication of SGIV in vitro. Both EcTRADD and EcTRADD-DD induced the caspase-dependent apoptosis in control and infected cells, while EcTRADD-N inhibited the apoptosis. Additionally, EcTRADD and EcTRADD-DD significantly promoted activation of NF-κB and reporter gene p53, whereas EcTRADD-N had no significant effect on p53. The results may provide new insights into the role of fish TRADD in fish virus infection.