EXOC8 of Epinephelus coioides involved in SGIV infection via innate immunity and apoptosis.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (Epinephelus coioides) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker (Larimichthys crocea) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro. Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.

Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, family Iridoviridae, frequently causes a severe disease with high mortality in grouper aquaculture. Although previous findings have demonstrated that SGIV envelope protein VP088 was crucial for its infectivity, the underlying mechanism still remained uncertain. Here, we screened the potential viral proteins that interacted with VP088 during SGIV infection using GFP pull-down assay. Co-immunoprecipitation (Co-IP) assays verified the interactions between VP088 and VP018, VP068, or VP156 in vitro. Moreover, confocal microscopy analysis showed that VP088 markedly altered the cellular distribution of exogenously expressed VP018 and VP068 and ultimately translocated into virus assembly sites together upon SGIV infection. Differently, VP088 mostly co-localized with exogenous VP156 in co-transfected cells and almost simultaneously translocated into the virus assembly sites, suggesting that VP088 participated in SGIV replication through interactions with other viral proteins in different ways. Interestingly, VP088 also abrogated IFN response induced by grouper (Epinephelus coioides) EccGAS-EcSTING and EcTBK1 in vitro. Co-IP assays showed that VP088 interacted with EccGAS, EcSTING, EcTBK1, or EcIRF3, while only degraded EcTBK1 via Ecp62-mediated autophagic degradation. Furthermore, VP088 decreased EcTBK1-induced EcIRF3 phosphorylation and nuclear translocation. In addition, the ectopic expression of VP088 attenuated the antiviral function of EcSTING/EcTBK1/EcIRF3 against red-spotted grouper nervous necrosis virus (RGNNV) infection. Thus, our results not only identified the association between SGIV VP088 and other viral proteins during replication, but also for the first time demonstrated that an iridoviral envelope protein could function as an immune evasion protein via abrogating EcTBK1-induced interferon response.IMPORTANCEIridovirus infection frequently causes high levels of morbidity and mortality among commercially and ecologically important fish, crustaceans, amphibians, and reptiles. However, the molecular mechanism of iridovirus pathogenesis still remains largely unknown, and few effective countermeasures have been developed to date. Using the Singapore grouper iridovirus (SGIV) infection model in vitro, we identified the potential viral proteins that interacted with envelope protein VP088 during virus replication. Moreover, for the first time, we demonstrated that VP088 interacted with EccGAS, EcSTING, EcTBK1, and EcIRF3, but only degraded EcTBK1 via Ecp62-mediated autophagic degradation, thereby inhibiting the host IFN response. Thus, our results not only contribute to elucidating the mechanism of SGIV pathogenesis but also provide a novel molecular target for the construction of immunogenic live vaccines against iridoviral diseases in the future.

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-β (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.

Singapore grouper iridovirus infection counteracts poly I:C induced antiviral immune response in vitro

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming

In mammals, mature miR-122 is 22 nucleotides long and can be involved in regulating a variety of physiological and biological pathways. In this study, the expression profile and effects of grouper Epinephelus coioides miR-122 response to Singapore grouper iridovirus (SGIV) infection were investigated. The sequences of mature microRNAs (miRNAs) from different organisms are highly conserved, and miR-122 from E. coioides exhibits high similarity to that from mammals and other fish. The expression of miR-122 was up-regulated during SGIV infection. Up-regulation of miR-122 could significantly enhance the cytopathic effects (CPE) induced by SGIV, the transcription levels of viral genes (MCP, VP19, LITAF and ICP18), and viral replication; reduce the expression of inflammatory factors (TNF-a, IL-6, and IL-8), and the activity of AP-1 and NF-κB, and miR-122 can bind the target gene p38α MAPK to regulate the SGIV-induced cell apoptosis and the protease activity of caspase-3. The results indicated that SGIV infection can up-regulate the expression of E. coioides miR-122, and up-regulation of miR-122 can affect the activation of inflammatory factors, the activity of AP-1 and NF-κB, and cell apoptosis to regulate viral replication and proliferation.

Expression and subcellular analyses of CCR8a/b genes with the identification of response to SGIV viral infect in orange-spotted grouper (Epinephelus coioides)

Characterization of p38 MAPKs from orange-spotted grouper, Epinephelus coioides involved in SGIV infection

Epinephelus coioides Hsp27 negatively regulates innate immune response and apoptosis induced by Singapore grouper iridovirus (SGIV) infection

Characterization of scavenger receptor MARCO in orange-spotted grouper, Epinephelus coioides

Characterization and functional analysis of GSK3β from Epinephelus coioides in Singapore grouper iridovirus infection

Comparative transcriptomic analysis reveals different host cell responses to Singapore grouper iridovirus and red-spotted grouper nervous necrosis virus

PPAR-δ of orange-spotted grouper exerts antiviral activity against fish virus and regulates interferon signaling and inflammatory factors

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

Antiviral function of grouper MDA5 against iridovirus and nodavirus.