Grouper spleen (GS) cell line from grouper, Epinephelus akaara, was established in our laboratory (33). GS cells were cultured in Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS, Gibco) at 25°C (33). Singapore grouper iridovirus (SGIV, strain A3/12/98 PPD) was propagated in GS cells and stored at −80°C until use (34).

Cellular toxicity detection of palmitic acid incubation was performed as described (35). Briefly, 100 mM palmitic acid (Sigma-Aldrich, St Louis, MO) stocks were prepared in 0.1 M NaOH at 70°C and filter sterilized. Bovine serum albumin (BSA, certified fatty acid free, low endotoxin, Sigma) was dissolved in complete media to a final concentration of 1% (w/v) and sterilized using a 0.45 μm non-pyogenic filter. Palmitic acid was added to a final concentration of 0.2, 0.4, 0.6, and 0.8 mM as different treatment levels. Control media (carrier) contained NaOH and filtered acid-free bovine serum albumin (BSA, Sigma-Aldrich).

In order to explore the impacts of palmitic acid on cell autophagy, chloroquine diphosphate salt (CQ, Sigma-Aldrich, C6628) was used in cell treatment (36, 37). GS cells were pretreated with palmitic acid for 20 h. And then, CQ was added into the culture medium at the final concentration of 5 μM and co-incubated for another 4 h.

It has been reported that the biosynthesis of palmitate is catalyzed by fatty acid synthase (FAS) (38). If FAS affected SGIV replication, palmitic acid could also influence it. The FASN (GenBank Accession No.: FJ196231) siRNA was designed using Thermo Fisher online tool BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress/sort.do). GS cells were transfected with FASN siRNA (siFASN: 5′-GGGUUCAAGUCGUUGACCAGCCUAU-3′) or the same volume of negative control (NC) siRNA for 24 h, and then infected with SGIV for 24 h. Cytopathic effects (CPE) caused by SGIV infection were observed under a light microscope (Zeiss). The effects of FASN siRNA on the transcriptional levels of viral genes were evaluated by qRT-PCR.

WST-1 assay was performed to examine the impact of palmitic acid treatment on cell proliferation. In brief, cells cultured in 96-well plates were incubated with palmitic acid at indicated concentrations (0, 0.2, 0.4, 0.6, and 0.8 mM) for 24 h. Cells were washed with culture medium three times. Then, 100 μL of culture medium was filled into each well. After adding 10 μL of cell proliferation reagent WST-1 (Roche) into each well and incubation at 28°C for 4 h, the absorbance was measured in Varioskan™ LUX multimode microplate reader (Thermo Fisher, USA) at 450/655 nm.

GS cells were seeded in 24-well plates for 18 h and then incubated with palmitic acid for 24 h. After that, cells were infected with SGIV at multiplicity of infection (MOI) of 0.5. The CPE was observed under a light microscope (Zeiss). Mock- or virus-infected cells were collected for further qRT-PCR analysis, western blot, and virus titer assay.

GS cells were infected with SGIV at MOI of 0.5. The total RNAs of cells were extracted using the SV Total RNA Isolation Kit (Promega) and reversed to synthesize the first-strand cDNA using the ReverTra Ace kit (Toyobo). Then the mRNA transcriptional levels of FA synthesis related genes were evaluated by qRT-PCR at 12 h post-infection (p.i.). The primers of ACC1 (Acetyl-CoA carboxylase 1), SREBP-1 (sterol regulatory element-binding protein 1), LXR (liver X receptor), and FASN gene (fatty acid synthase) are listed in Table 1.

In order to determine the roles of palmitic acid in virus infection, GS cells were incubated with palmitic acid for 24 h, and infected with SGIV for 24 h at 28°C. Virus-infected cells were collected for RNA extraction and qRT-PCR analysis. The qRT-PCR analysis was performed in the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher, USA). Each assay was carried out in triplicate. The mRNA expression level of viral genes, including SGIV-MCP (major capsid protein), SGIV-VP19, SGIV-ICP-18, and SGIV-LITAF (lipopolysaccharide-induced TNF-α factor) as well as the host genes, were evaluated and the primers are listed in Table 1. The data were calculated as the folds based on the expression level of targeted genes normalized to β-actin.

Viral replication kinetics was assessed in GS cells to determine the effect of palmitic acid on SGIV production. In brief, GS cells pretreated with palmitic acid or vehicle for 24 h were infected with SGIV (at MOI of 0.5) and collected at 48 h p.i. for virus titer determination. The viral titers of cell lysates were evaluated using the 50% tissue culture infectious dose (TCID₅₀) assay (39). The CPEs were observed under a light microscope (Leica, Germany) every day, and each sample was measured in triplicate.

After the experimental treatments, cells were lysed and solubilized in 40 μL of Pierce IP Lysis Buffer (Thermo Fisher Scientific), containing protease/phosphatase inhibitor cocktail. Samples were boiled for 5 min after mixing with 5× loading buffer. Solubilized proteins were resolved by 6, 10, or 12% SDS-PAGE and then electrophoretically transferred to 0.2 μm Trans-Blot Turbo PVDF (Minipore). The membranes were blocked with 5% skim milk or 3% bovine serum albumin (BSA) dissolved in PBS for 2 h, then incubated with different primary antibodies overnight at 4°C. Membranes were washed for 3 times in PBST or TBST (for phosphorylation assay) buffer subsequently. Then, secondary goat-anti-rabbit or goat-anti-mouse antibody labeled with horseradish per-oxidase was used, and bound proteins were detected with Enhanced HRP-DAB chromogenic substrate Kit (TIANGEN; www.tiangen.com) according to the manufacturer's protocol. The following primary antibodies were used: anti-LC3B (1:1,000 dilution, Abcam), anti-p62 (1:1,000 dilution, Abcam), anti-Akt (1:1,000 dilution, Abcam), anti-p-Akt (Ser473) (1:1,000 dilution, Cell Signaling), anti-mTOR (1:1,000 dilution, Cell Signaling), anti-p-mTOR (1:1,000 dilution, Abcam), anti-SGIV-MCP (1:1,000 dilution), and anti-β-tubulin (1:5,000 dilution, Abcam). Data were normalized to the mean of β-tubulin expression.

Applied as a vital stain for the detection of intracellular lipid droplets, Nile Red (9-diethylamino-5H-benzo[α] phenoxazine-5-one) is characterized by red fluorescence (excitation 515–560 nm, emission >590 nm) (40, 41). The stock solution of Nile Red was prepared in acetone (0.5 mg/mL) and stored at 4°C, protected from light. After 24 h incubation with palmitic acid, GS cells were washed three times with phosphate buffer saline (PBS). After that, the cells were fixed with 4% paraformaldehyde for 30 min and then stained with Nile Red solution in the dark for 5 min at room temperature (42). The stain of intracellular lipid in GS cells was performed using a solution of Nile Red (5 μg/mL) by diluting the stock solution of dye (1:100) in PBS (41). Afterward, the cells were washed with PBS three times. The GS cells' steatosis was visualized using fluorescence microscopy (Zeiss, Germany), and the intracellular lipid vacuoles showed red fluorescence. The fluorescence intensity analysis of intracellular fat accumulation in GS cells was performed by Image J (https://imagej.en.softonic.com/). Each experiment was repeated three times.

Virus-infected GS cells or mock cells were fixed with 4% paraformaldehyde at 24 h p.i. After incubation with anti-MCP (SGIV) (1:100), cells were washed with PBS and incubated with FITC-conjugated goat anti-rabbit antibodies (Pierce). Then they were stained with Nile Red as described previously. Finally, samples were stained with 1 mg/mL 6-diamidino-2-pheny-lindole (DAPI) and observed under fluorescence microscopy (Zeiss, Germany).

Palmitoylation is a type of post-translational modification and occurs when fatty acids like palmitic acid, are covalently attached to side chains of cysteine in proteins. Here, EZClick™ Palmitoylated Protein Assay Kit (Biovision, Catalog # K452-100) was used to detect the influence of SGIV infection on the cellular palmitic acid level. According to the protocol, Negative Control Cells are unstained cells, and cells are not exposed to Palmitic Acid Label or EZClick™ Fluorescent Azide. Background Control Cells are only exposed to EZClick™ Reaction, no EZClick™ Palmitic Acid Label. Positive Control Cells are incubated with 1× EZClick™ Palmitic Acid Label and EZClick™ Reaction. Experimental Cells are incubated with SGIV and then exposed to EZClick™ Reaction. Briefly, GS cells were seeded in 24-well plate overnight, then cells of Experimental Group were infected with SGIV (MOI = 0.5) for 12 h. For the positive control cells, media was replaced with fresh aliquots containing EZClick™ Palmitic Acid Label (1,000×) diluted to 1× final concentration with the culture medium. After 12 h incubation, 1× PBS solution was used to terminate the experiment. Then, cells were incubated with Fixative Solution for 15 min at room temperature, protected from light. After fixation, 1× PBS was used to wash the cells. Then 1× Permeabilization Buffer was added to the cells for 10 min incubation at RT. For Negative Control Cells, 400 μL of 1× PBS were added. For Background Control Cells, Positive Control Cells, and Experimental Cells, 400 μL of EZClick™ Reaction Cocktail were added, and then they were incubated for 30 min at RT protected from light. After that, the Reaction Cocktail or PBS was removed, and the cells were washed with 1× Wash Buffer three times. After staining with Hoechst33342, the cells were washed with 500 μL of ice-cold PBS and analyzed through fluorescence observation.

The effects of the palmitic acid on the promoter activity of zebrafish interferon 1 (IFN1) and interferon sequence response element (ISRE) were evaluated by reporter gene assays (43). In brief, GS cells were cultured in 24-well plates before co-transfecting with 0.8 mg ISRE-Luc/IFN1-Luc and 0.05 mg pRL-SV40 Renilla luciferase vector. At 24 h post-transfection, cells were incubated with palmitic acid, then collected at 48 h post-transfection. Luciferase activity in total cell lysates was measured by luciferase reporter assay (Promega, USA) using a Varioskan™ LUX multimode microplate reader (Thermo Fisher, USA). Co-transfection assay was carried out as described previously by using pcDNA-flag-EcIRF3/EcIRF7/EcTBK1/EcMDA5.

Results were expressed as means±SD. Statistical comparisons were conducted using the Student's t-test, and a p-value of < 0.05 was considered to be statistically significant.

To demonstrate whether fatty acids were associated with SGIV infection in GS cells, we first detected the changes of fatty acid synthesis after SGIV infection. The evaluation of palmitoylated protein staining suggested the lipid synthesis was promoted by SGIV infection in GS cells (Figure 1A). Virus infection increased the accumulation of lipid products, especially palmitic acid, in GS cells. As shown in Figure 1A, there was strong green fluorescent signal in the cytoplasm of positive control cells, but no signal in negative control ones. In SGIV infected cells, green fluorescence was observed in cytoplasm, which suggested that palmitoylated protein was accumulated (Figure 1A). Figure 1B shows that besides palmitic acid, SGIV infection could increase the production of other intracellular lipids. Our previous study on lipid metabolic profile of SGIV infected cells also suggested SGIV infection stimulated the accumulation of palmitic acid and at least two other fatty acids, oleic acid and eicosatetraenoic acid (unpublished data). Thus, we speculated that SGIV could utilize the lipid synthesis systems and control the cellular palmitoylation for its replication.

Genes correlated with lipids played essential roles in regulating the metabolism of lipids and participated in virus infection (44). The up-regulated levels of fatty acid regulating genes due to SGIV infection indicated that these genes could have a positive effect on SGIV infection (Figure 1C). Functioning as the catalyst in palmitate biosynthesis processes (38, 45, 46), FAS was also induced by SGIV in GS cells. Then, RNA interfering assay was carried out to knock down the fatty acid synthase gene (FASN) (Figure 2A), leading to inhibition in virus replication of SGIV (Figures 2B,C). The CPE of SGIV infection was less severe in FASN knock down cells (Figure 2B). Similarly, the mRNA level of SGIV functional genes was reduced in FASN siRNA transfection group (Figure 2C). These results suggested FASN gene was essential for SGIV replication. An earlier report had revealed that fatty acid synthase (FAS) encoded by FASN gene was a multi-functional enzyme that catalyzed palmitate biosynthesis in a NADPH-dependent reaction (38). However, the mechanisms of palmitate in modulating SGIV infection and replication remain unclear. Functional analysis should be carried out to investigate the role of palmitic acid in host-virus interaction.

Changes of metabolic profile of GS cells in response to SGIV infection were documented. Palmitic acid massively increased in SGIV infected cells (unpublished data). To investigate the potential functions of palmitic acid in SGIV infection, we utilized an in vitro model in which GS cells were loaded with palmitic acid as previously described (3, 35). Palmitic acid showed no adverse effect on cell viability when the concentration was lower than 0.6 mM within 24 h incubation (Figure 3A). But 0.6 mM palmitic acid was toxic when incubating for 72 h or more (data no shown). A microscopic evaluation of GS cells after incubation with palmitic acid showed that the cytoplasm of palmitic acid pretreated cells contained numerous different-sized fluorescent bodies (stained with Nile Red) corresponding to lipid accumulation, i.e., steatosis (Figure 3B). In the cytoplasm of control cells (1% BSA), the presence of moderate micro- and macro-vacuolar steatosis was also observed (Figure 3B).

To investigate the effects of palmitic acid on SGIV virus infection, we evaluated the CPE progression and detected the viral gene transcription as well as the viral coat protein synthesis of SGIV in infected palmitic acid loading cells. As shown in Figure 4, SGIV infection induced CPE was more severe in palmitic acid treated cell groups than in untreated cells (Figure 4A). Moreover, in palmitic acid loaded cells, the transcription level of SGIV-MCP, SGIV-ICP-18, SGIV-VP19, and SGIV-LITAF was significantly increased (Figure 4B). And the synthesis of SGIV MCP detected by western-blotting also showed that palmitic acid improved the expression of SGIV major coat protein (Figure 4C). Virus titer assay showed increased viral replication of SGIV after palmitic acid treatment (Figure 4D). The results of virus titer assay also suggested that exogenous addition of palmitic acid enhanced SGIV replication (Figure 4D).

It has been demonstrated that palmitic acid can modulate autophagy in many kinds of cells. We sought to determine whether palmitic acid could also modify this process in GS cell (5, 47). Firstly, the green fluorescence of LC3 was observed under a fluorescence microscope with and without palmitic acid treatment. As shown in Figure 5, increased levels of green signal of LC3, as well as the higher levels of LC3-II and p62 in the palmitic acid treated group evidenced by western blot assay indicate that palmitic acid induces the accumulation of autophagosomes in GS cells (Figures 5A,C). Autophagy is known to be a dynamic process, so the detection of LC3-II levels is not sufficient to assess autophagic activity in cells (5, 48). The inhibition of palmitic acid on p-Akt (Ser473) and p-mTOR was evaluated (Figure 5B). It has been highlighted that the reduced level of p-Akt and p-mTOR could contribute to the promotion of autophagy (49). However, the increased level of LC3-II may be due to the increased autophagic activity, or the blockades of lysosomal function (5, 48). To discriminate between these two possibilities, we determined the impacts of palmitic acid on autophagic flux using chloroquine (CQ), an inhibitor of autophagosome-lysosome fusion. GS cells were incubated with vehicle (1% BSA), 0.2 mM palmitic acid, or 0.4 mM palmitic acid for 24 h, and CQ was added for the last 4 h treatment. The pixel values of LC3-II and β-tubulin were evaluated using Image J. Then, the autophagic flux was measured as the ratio of LC3-II level (LC3-II/β-tubulin) in cells treated with CQ to that of untreated cells as described previously (5). As shown in Figure 5E, the calculated result was performed in histogram. The autophagic flux was blocked in the presence of palmitic acid (Figure 5E). Taken together the decreased autophagic flux and the accumulation of LC3-II and p62 caused by palmitic acid, we subsequently concluded that palmitic acid decreased autophagy flux by inhibition of autophagosome–lysosome fusion step.

To determine the regulatory effects of palmitic acid on the expression of interferon related molecules, we detected the transcripts of these genes in GS cells treated with different concentrations of palmitic acid. As shown in Figure 6, the relative expression of EcIRF3 (interferon regulatory factors 3), EcIRF7, EcISG15 (interferon-stimulated gene), EcIFP35 (Interferon-induced 35 kDa protein), EcMXI (Interferon-induced GTP-binding protein Mx1), EcTBK1 (TANK-binding kinase 1), EcTRIF (TIR-domain-containing adapter-inducing interferon-β), and EcMDA5 (melanoma differentiation-associated protein 5) were significantly decreased in palmitic acid treated cells compared to the control cells, suggesting that palmitic acid inhibited the interferon responses (Figure 6).

On the other hand, the effects of palmitic acid treatment on IFN and ISRE promoter activities were evaluated using reporter gene assay. Palmitic acid treatment not only decreased the IFN and ISRE promoter activities (Figure 7A) but also suppressed IRF3/IRF7 evoking interferon signaling pathway (Figure 7B).

Furthermore, the insights of palmitic acid modulations were discovered by detecting its regulation of MDA5-/TBK1-induced interferon signaling pathway. Transcriptional levels of EcIRF3, EcIRF7, and EcISG15 were detected using qRT-PCR method. As shown in Figure 8, palmitic acid significantly decreased the TBK1-induced interferon responses. But there were no significant differences between BSA or palmitic acid incubated groups under MDA5 transfection. Thus, we proposed that palmitic acid treatment could down-regulate IFN signaling by inhibiting the TBK1-IRF3/7 pathway.