Juvenile grouper (40-50 g in weight) were obtained from the fishery in Yangjiang City, China. They were stored in a recirculating seawater system at 24-28°C and fed twice daily for two weeks. Then three groupers were randomly selected to detect whether the fish was infected with bacteria or viruses. Twelve tissues were extracted from 6 healthy fish, immediately frozen in liquid nitrogen, and stored at -80°C.

Grouper spleen (GS) cells were constructed in our laboratory and are currently kept in our laboratory. GS cells were grown in Leibovitz L15 medium (Wibco, Waltham, MA, USA) containing 10% fetal bovine serum and placed in a 28°C incubator (14, 15). SGIV was isolated from diseased groupers and cultured as previously described (1, 16). Viral cultures were maintained at -80°C.

Rabbit monoclonal anti-green fluorescent protein (GFP) antibody was purchased from Sigma (Burlington, MA, USA), and mouse monoclonal anti-HA antibody was also purchased from Sigma (Burlington, MA, USA). Rabbit monoclonal β-tubulin antibody was purchased from Proteintech (Rosemont, IL, USA). Polyclonal antibody to SGIV protein MCP was prepared in our laboratory. Horseradish peroxidase-labeled goat anti-rabbit antibody as secondary antibody was purchased from KPL (USA).

Primers used to amplify the open reading frame (ORF) of EccGAS were designed according to the EST sequences of cGAS in the grouper spleen transcriptome (1, 16). The ORF of EccGAS was amplified from the tissue cDNA of healthy grouper. Sequences of EccGAS were analyzed using the BLAST program (http://www.ncbi.nlm.nih.gov/blast), and the conservative domains were predicted using the conservative domain database (https://www.ncbi.nlm.nih.gov/cdd/) of NCBI. SignalP 4.1 was used to predict signal peptides and TMHMM Server V. 2.0 was used to predict transmembrane regions. GeneDoc and Clustal X1.83 were used for amino acid sequences alignment of cGAS, and MEGA version 6.0 was used for phylogenetic tree analysis.

Total RNA was performed using the SV Total RNA Isolation System (Promega, United States) following the manufacturer’s instructions. cDNA synthesis was performed with the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) according to the manufacturers’ instructions. SYBR^® Green Real-Time PCR Master Mix (Toyobo) was used to perform the quantitative real-time PCR (qRT-PCR) in an Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo Fisher, Waltham, MA, USA), as previously described (17). Briefly, each assay was performed in triplicate with the cycling conditions as follows: 95°C for 1 min for activation, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. The primers of target genes are listed in Table 1 , and β-actin was used as the internal reference gene. The expression levels were calculated using the 2^–ΔΔCT method.

Primers were designed to amplify the ORF of EccGAS ( Table 1 ). The PCR product was digested with BamH I and EcoR I (Takara) and subsequently subcloned into the expression vector pET-B2M. Positive clones were incubated at 37°C, and shaked at 220 rpm in a 150 mL LB medium with 100 mg/mL ampicillin. The vector pET-B2M was used as a negative control. When OD600 of the medium reached 0.6, IPTG inducer (final concentration 0.5 mM) was added, and the culture was shaken at 37°C for 3 h. Recombinant EccGAS fusion proteins (named rEccGAS) were purified. The concentration of purified rEccGAS protein was determined via Bradford’s method (18). The purified rEccGAS proteins were then used to immunize the New Zealand white rabbits to obtain polyclonal antibodies against EccGAS according to conventional methods (19). Western blot was used to detect the specificity of the antiserum.

Cell transfection was performed with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, cells were seeded into plates and changed to serum-free medium after the cell density had spread to 80%. Lipofectamine 2000 and plasmids were diluted with Opti-MEM (Gibco, USA) in two separate sterile tubes. After a 5-min incubation at 25°C, Lipofectamine 2000 with the diluted plasmids were mixed gently and thoroughly. The mixture was then incubated at 25°C for 25 min before being added dropwise to the cells. After 5-6 h, the medium was changed to serum medium to continue the culture (20).

GS cells were seeded into 6-well plates containing coverslips (10 mm×10 mm). When the cell density is appropriate, the plasmids are transfected. After 24 h, cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde or methanol for 1 h. Cells were permeabilized with anhydrous ethanol for 15 min at -20°C and then blocked with 2% BAS (ready-to-use) for 2 h at 25°C. Cells were incubated with 1% BSA diluted with rabbit polyclonal anti-EccGAS antibody (1:150) or mouse monoclonal anti-HA antibody (1:150) for 2 h. Cells were incubated for 2 h with 1% BSA diluted with rabbit polyclonal anti-EccGAS antibody (1:150) or mouse monoclonal anti-HA antibody (1:150). Cells were then washed with phosphate-buffered saline. FITC-conjugated goat anti-rabbit or goat anti-mouse antibodies were diluted (1:200) with 1% BSA and incubated for 1 h. Cells were then washed with phosphate-buffered saline and treated with 6-dibutylamino-2-phenylindole (DAPI) for 10 min in the dark, and observed under a fluorescence microscope (Leica, Wetzlar, Germany).

Three siRNAs targeting EccGAS mRNA were designed to evaluate the mechanism of EccGAS on SGIV infection in GS cells ( Table 2 ). Cells were transfected with the same volume of siRNA or control. The cells were infected with SGIV 24 hours later. The cells were then collected at 24 h and 36 h after SGIV infection. The expression levels of SGIV ICP18, VP19, and MCP were analyzed using qRT-PCR ( Table 1 ). The expression of SGIV MCP protein was analyzed using western blotting.

GS cells were plated in 24-well plates. When the cell density is appropriate, a total of 200 ng of zebrafish IFN1-LUc, human NF-κB-Luc, or human ISRE-Luc plasmids were co-transfected with 30 ng of the internal control PRL-sv40 reninase vectors and 600 ng of the target plasmids. After 36 h, cells were collected, and the luciferase activity was detect by the Dual-Luciferase^® Reporter Assay System (Promega).

GS cells were passaged into cell culture dishes (10cm×10cm). When the cell density is appropriate, co-transfected with relevant the target plasmids. After 36 h, cells were harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. Samples were processed using the Double Bead™ Protein G Immunoprecipitation Kit (Invitrogen).

10% SDS-PAGE was used to separate immunoprecipitates or whole cell extracts. Separated immunoprecipitates or whole cell extracts were then transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, St. Louis, MO, USA). The membranes were blocked in 5% skim milk incubated with antibodies for 2 h at 25°C or overnight at 4 °C, washed 3 times with PBST, and incubated with secondary antibodies for 1 h at 25°C. After washing three times with PBST, the immunoreactive protein was visualized by an enhanced chemiluminescence detection kit (Bio-Rad, Irvine, CA, USA). The band intensity was calculated using Quantity-one software (21).

GraphPad Prism (version 8.0.2) was used to perform statistical analysis. Data analysis results are shown as mean ± standard error of the mean of three independent experiments. The statistically significant differences were evaluated by the T-Test with a P value (*P < 0.05 and **P < 0.01).

The ORF of grouper cGAS (EccGAS) was 1695 bp in length, encoding 575 amino acids, with a molecular weight of approximately 63.05 kDa and an isoelectric point of 9.47. SMART analysis revealed that EccGAS contains the Mab-21 typical structural domain, while the signal peptide and transmembrane region were absent.

Aligning the cGAS proteins of other organisms with EccGAS revealed that the sequences of each species were relatively conserved in the Mab-21 typical structural domain, indicating that cGAS may have similar functions. The cGAS sequences of 11 species were selected for homology comparison, and the multiple sequence alignments were performed with Clustal X ( Figure 1A ). The phylogenetic tree showed that the EccGAS and cGAS of Sebastes umbrosus (XP_037637334.1) were more closely related and clustered together ( Figure 1B ).

The expression of EccGAS in healthy grouper tissues showed that EccGAS was expressed in all exacted tissues, with higher expression in blood, skin, and gills ( Figure 2A ). After SGIV infection, the transcriptional expression level of EccGAS in GS cells gradually increased and peaked at 36 h.p.i ( Figure 2B ), suggesting that EccGAS may play an important role in the activation of host antiviral innate immunity.

The recombinant protein of EccGAS was analyzed by SDS-PAGE. A band of 57 kDa was visible using Komas Brilliant Blue staining ( Figure 3A ). The fused recombinant EccGAS protein (rEccGAS) was purified by affinity chromatography with nickel-nitrilotriacetic acid-agarose (QIAGEN, Germany) according to the manufacturer’s instructions ( Figure 3B ). Immunosera were then prepared by immunizing rabbits with rEccGAS protein. rEccGAS protein was specifically recognized by the EccGAS polyclonal antibody, and no bands were detected in the negative control ( Figure 3C ), indicating that the anti-EccGAS antibody specifically recognized the purified EccGAS protein.

The expression vectors for full-length EccGAS, structural domain Mab21, and removal of the structural domain were constructed and named EccGAS(EccGAS), EccGAS-Mab21, and EccGAS-delete-mab21. Subsequently, the plasmids were transfected into GS cells, and intracellular localization of EccGAS was determined. The results showed that the green fluorescence of pEGFP-C1 was distributed in the cytoplasm and nucleus of GS cells, while the pEGFP-EccGAS and pEGFP-EccGAS-delete-mab21 were distributed uniformly in the cytoplasm, and pEGFP-EccGAS-Mab21 was distributed in the cytoplasm in an aggregated manner ( Figure 4A ). We next examined the intracellular localization of EccGAS in GS cells by immunofluorescence assay with anti-EccGAS serum. In GS cells, the green and red fluorescences of EccGAS were mainly localized in the cytoplasm by anti-EccGAS serum ( Figure 4B ), which is consistent with the results of subcellular localization.

To explore whether EccGAS co-localizes with organelles, plasmids pEGFP-EccGAS were co-transfected with pDsRed2-ER (endoplasmic reticulum) or pDsRed2-Mito (mitochondria) into GS cells and their localization was examined. The results showed that EccGAS partially co-localized with the endoplasmic reticulum and mitochondria ( Figure 4C ).

We designed three siRNAs based on the ORF sequence of EccGAS. siRNAs were transfected into GS cells, and qRT-PCR was used to detect the expression of endogenous EccGAS after 24 hours. The results showed that all three siRNAs silenced the expression of endogenous EccGAS in GS cells, of which siRNA2 had the highest interference efficiency ( Figure 5A ). Next, we used siRNA2 to conduct the following experiments. After siRNA2 was transfected into GS cells and infected with SGIV, the expression of SGIV MCP, SGIV ICP18, and SGIV VP19 were examined by qRT-PCR. The expression of SGIV MCP protein was detected by western blot. The results showed that EccGAS knockdown by siRNA2 significantly inhibited the transcript expression of SGIV genes ( Figure 5B ). Moreover, the results of western blot showed that knockdown EccGAS by siRNA2 significantly reduced the expression of MCP protein ( Figure 5C ). These results suggest that EccGAS may promote SGIV replication in GS cells.

The regulatory effects of EccGAS on host immune factors were evaluated by qRT-PCR. As shown in Figure 6 , EccGAS knockdown by siRNA2 potentiated the transcription of IFN1, IFN2, PKR, Viperin, ISG15, ISG56, IL-1β, IL-8, and TNFα in GS cells. Thus, the results suggested that EccGAS negatively regulated the interferon immune response in vitro.

In mammals, cGAS has been reported to produce cGAMP after DNA sensing to stimulate STING, which in turn activates interferon production and innate immunity. We aimed to determine whether EccGAS is involved in EcSTING-mediated interferon immune response. As shown in Figure 7A , when EccGAS was co-transfected with EcSTING, EcTBK1, EcTAK1, and EcIRF3, the activities of IFN1, ISRE, and NF-κB promoters were significantly reduced, suggesting that EccGAS may be involved in EcSTING-mediated interferon immune responses. Next we investigated whether EccGAS interacts with EcSTING, EcTBK1, EcTAK1 and EcIRF3. Confocal microscopy results showed that the green fluorescence of pEGFP-EcSTING, pEGFP-EcTBK1, pEGFP-EcTAK1, and pEGFP-EcIRF3 colocalized with the red fluorescence of EccGAS ( Figure 7B ). We further verified the experimental results by CO-IP assay. The plamids of pcDNA3.1-EccGAS was co-transfected with pEGFP-EcSTING, pEGFP-EcTBK1, pEGFP-EcTAK1, pEGFP-EcIRF3, and pEGFP-C1, respectively, followed by immunoprecipitation (IP) and immunoblotting (IB) of whole cell lysates (WCLs). The results showed that EccGAS was detected in the IP products of EcSTING, EcTAK1, EcTBK1, and EcIRF3, but not in pEGFP-C1 ( Figures 7C–E ). Therefore, EccGAS inhibited EcSTING-mediated interferon immune response and interacted with EcSTING, EcTAK1, EcTBK1, and EcIRF3.