Functional Analysis of the Cathepsin D Gene Response to SGIV Infection in the Orange-Spotted Grouper, Epinephelus coioides.

(1) Background: Lysosomal aspartic protease Cathepsin D (CD) is a key regulator and signaling molecule in various biological processes including activation and degradation of intracellular proteins, the antigen process and programmed cell death. However, the function of fish CD in virus infection remains largely unknown. (2) Methods: The functions of the CD gene response to SGIV infection was determined with light microscopy, reverse transcription quantitative PCR, Western blot and flow cytometry. (3) Results: In this study, Ec-Cathepsin D (Ec-CD) was cloned and identified from the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of Ec-CD consisted of 1191 nucleotides encoding a 396 amino acid protein with a predicted molecular mass of 43.17 kDa. Ec-CD possessed typical CD structural features including an N-terminal signal peptide, a propeptide region and a mature domain including two glycosylation sites and two active sites, which were conserved in other CD sequences. Ec-CD was predominantly expressed in the spleen and kidneys of healthy groupers. A subcellular localization assay indicated that Ec-CD was mainly distributed in the cytoplasm. Ec-CD expression was suppressed by SGIV stimulation and Ec-CD-overexpressing inhibited SGIV replication, SGIV-induced apoptosis, caspase 3/8/9 activity and the activation of reporter gene p53 and activating protein-1 (AP-1) in vitro. Simultaneously, Ec-CD overexpression obviously restrained the activated mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). In addition, Ec-CD overexpression negatively regulated the transcription level of pro-inflammatory cytokines and activation of the NF-κB promotor. (4) Conclusions: Our findings revealed that the Ec-CD possibly served a function during SGIV infection.

High-density lipoproteins negatively regulate innate immunity and facilitate red-spotted grouper nervous necrosis virus entry via scavenger receptor B type 1.

Lipid metabolism plays an important role in viral infections, and it can directly or indirectly affect various stages of viral infection in cells. As an important component of lipid metabolism, high-density lipoprotein (HDL) plays crucial roles in inflammation, immunity, and viral infections. Scavenger receptor B type 1 (SR-B1), a receptor of HDL, cannot be ignored in the regulation of lipid metabolism. Here, we investigate, for the first time, the role of Epinephelus coioides SR-B1 (Ec-SR-B1) in red-spotted grouper nervous necrosis virus (RGNNV) infection. Our results indicate that Ec-SR-B1 could promote RGNNV infection. We also demonstrate that Ec-SR-B1 could facilitate viral entry and interact with capsid protein (CP) of RGNNV. As the natural ligand of SR-B1, HDL significantly increased RGNNV entry in a dose-dependent manner. However, we observed no effect of HDL on Ec-SR-B1 expression. The results of the micro-scale thermophoresis assay did not reveal an association between HDL and CP, suggesting that RGNNV does not enter target cells by using HDL as a ligand to bind to its receptor. In addition, block lipid transport-1, a compound that inhibits HDL-mediated cholesterol transfer, reduced the HDL-induced enhancement of RGNNV infection, indicating a role for lipid transfer in facilitating RGNNV entry. Furthermore, HDL inhibited the expression of pro-inflammatory factors and antiviral genes in a dose-dependent manner. These findings suggest that the HDL-induced enhancement of RGNNV entry involves the complex interplay between Ec-SR-B1, HDL, and RGNNV, as well as the regulation of innate antiviral responses by HDL. In summary, we highlight the crucial role of HDL in RGNNV entry, identify a possible molecular connection between RGNNV and lipoprotein metabolism, and indicate the role of Ec-SR-B1 in RGNNV infection.

Identification and functional characterization of Cystatin B in orange-spotted grouper, Epinephelus coioides.

Cystatin B is a cysteine protease inhibitor that plays a crucial role in immune response. Nevertheless, the molecular mechanism of fish Cystatin B in virus replication remains obscure. In this study, we identified and characterized Cystatin B (Ec-CysB) in the orange-spotted grouper (Epinephelus coioides). The Ec-CysB encoded a 100-amino acid protein with the conserved QXVXG motif, PC motif and cysteine protease inhibitory motif, which shared high identities with reported Cystatin B. The abundant transcriptional level of Ec-CysB was found in gill, intestine and head kidney. And the Ec-CysB expression was significantly up-regulated in spleen after infection with Singapore grouper iridovirus (SGIV) in vitro. Subcellular localization analysis revealed that Ec-CysB was distributed mainly in the cytoplasm and nucleus. Further studies showed that overexpression of Ec-CysB in vitro significantly increased SGIV replication and virus-induced cell apoptosis, but replication of SGIV was inhibited by knockdown or mutant of Ec-CysB. Moreover, overexpression of Ec-CysB significantly inhibited the interferon (IFN), interferon-stimulated response element (ISRE) promoter activities, and enhanced apoptosis-related transcription factors p53 promoter activities. Collectively, our results suggest that Ec-CysB affect viral replication and virus-induced cell apoptosis, which will help us to explore its potential functions during SGIV infection.

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

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor that plays a crucial role in the immune response against microbial infections. To clarify the roles of fish MARCO in Singapore grouper iridovirus (SGIV) infection, we identified and characterized Ec-MARCO in the orange-spotted grouper (Epinephelus coioides). The Ec-MARCO encoded a 370-amino acid protein with transmembrane region, coiled coil region and SR domain, which shared high identities with reported MARCO. The abundant transcriptional level of Ec-MARCO was found in spleen, head kidney and blood. And the Ec-MARCO expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-MARCO was mainly distributed in the cytoplasm and on the cell membrane. Ec-MARCO knockdown in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that Ec-MARCO knockdown positively regulated proinflammatory cytokines and interferon-stimulated genes, and enhanced IFN and ISRE promoter activities. However, overexpression of Ec-MARCO did not affect SGIV entry into host cells. In summary, our results suggested that Ec-MARCO affected SGIV infection by regulating antiviral innate immune response.