Mandarin fish (S. chuatsi), averaging 50 grams in weight, were sourced from a farm in Guangdong province, China. Prior to the experiment, the fish were acclimated for two weeks in a laboratory-controlled, recirculating freshwater system at 27°C. All animal procedures were conducted in accordance with Guangdong Province’s animal experimentation regulations and approved by the ethics committee of Sun Yat-sen University. The mandarin fish fry (MFF-1) cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, USA), supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, USA), and maintained at 27°C in a humidified atmosphere with 5% CO₂ (30). Fathead minnow (FHM) cells (ATCC CCL-42) were maintained in M199 medium supplemented with 10% FBS (Gibco, Grand Island, USA) at 27°C (31). The mandarin ranavirus (MRV) strain NH-1609 (Accession number: MG941005) was isolated from moribund hybrid mandarin fish in Naihai, Guangdong, China, and is preserved in our laboratory (31).

Mouse monoclonal anti-c-Myc antibody and mouse monoclonal anti-Flag antibody were obtained from Sigma–Aldrich (St. Louis, USA). The anti-Ha tag antibody was purchased from Cell Signaling Technology (CST, Danvers, USA). Anti-GAPDH was obtained from Abways (Shanghai, China), and rabbit polyclonal anti-mrvORF097L antibody was produced in our laboratory (32). Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) and HRP-conjugated goat anti-rabbit IgG (H+L), both supplied by Promega (Madison, USA). The proteasome inhibitor MG132, cycloheximide (CHX), 3-Methyladenine (3-MA), and chloroquine (CQ) were all purchased from MedChemExpress (New Jersey, USA). Lodoacetamide (IAA) was obtained from CST (Danvers, USA).

For protein domain analysis, SMART (Simple Modular Architecture Research Tool, a web resource for the identification and annotation of protein domains and the analysis of protein domain architectures was used to compartmentalize the protein domains of scRNF5 in Genomic Mode (33). For evolutionary tree, protein sequences were retrieved from GenBank (34) and RefSeq (35) databases and aligned by ClustalW in MEGA v11.0.13 (a software contains a large collection of methods and tools of computational molecular evolution) (36). Then the evolutionary tree was constructed in MEGA using the Neighbor-Joining method with 1,000 bootstrap replications, Jones-Taylor-Thornton (JTT) model, and complete deletion. The evolutionary tree was visualized using iTOL (an online tool for the display, annotation and management of phylogenetic and other trees) (37), incorporating cartoon elements from the Generic Diagramming Platform (a comprehensive database of high-quality biomedical graphics) (38). For protein three-dimensional (3–D) structure predictions, protein sequences were obtained from RefSeq databases, and Alphafold Server (a web server can predict the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues) (39) was employed to predict the protein structures. For protein amino acid sequence alignment, protein sequences were obtained from GenBank and RefSeq databases and DNAMAN v9.0 (a one-for-all software package for molecular biology applications) was used to align with Dynamic Alignment and Default Parameters.

Total RNAs were extracted, and cDNAs were synthesized following previously described methods (25). scRNF5 and scSTING were constructed via polymerase chain reaction (PCR), cloned, and then inserted into pCMV-Myc-N and pCMV-Flag-N vectors (Takara, Japan). This process generated the plasmids: pCMV-Myc-scRNF5, pCMV-Flag-scSTING, pCMV-Flag-scSTING (1–140), pCMV-Flag-scSTING(141-417), pCMV-Flag-scSTING(1-180), and pCMV-Flag-scSTING(181-417) plasmids Various STING constructs, including Ha-scSTING(1-180)KR, Flag-scSTINGCKR, Flag-scSTINGCKRK20, Flag-scSTINGCKRK95, Flag-scSTINGCKRK117, Flag-scSTINGCKRK122, Flag-scSTINGCKRK135 and Flag-scSTINGCKRK155, were synthesized by Beijing Tsingke Biotechnology (China). The primers used for gene cloning, which contained restriction enzyme sites, are listed in Supplementary Material 1 . The plasmids were transfected into FHM cells using Fugene HD (Promega, USA), and into MFF-1 cells using Transfect EZ 3000 Plus (eLGbio, China), adhering to the manufacturers’ standard protocols.

To examine the tissue distribution of scRNF5 in healthy fish, total RNAs were extracted from various tissues including the gills, fins, spleen, intestine, brain, head kidney, hind kidney, middle kidney, blood, fat, heart, liver, and muscle. Extraction was performed using the SV Total RNA Isolation Kit (Promega, USA) following the manufacturer’s instructions. The expression levels of scRNF5 in these tissues were then quantified by quantitative reverse-transcription PCR (qRT-PCR) using the primers listed in Supplementary Material 1 .

MFF-1 cells were cultured in 24-well plates and co-transfected with pRL-TK and IFN-β-Luc plasmids (Promega, USA), and the indicated plasmids or empty vector. After 36 h, cells were harvested, lysed in 200 µL of Passive Lysis Buffer (Promega, USA), and subjected to the Dual-Luciferase Reporter Gene Assay Kit (Promega, USA) following the manufacturer’s instructions. Luciferase activities were measured using Glomax instrument (Promega, USA). Experiments were performed in triplicate, and data represented the mean of at least three independent experiments. All experiments were conducted in at least three independent trials with three technical replicates.

Total RNA was extracted from cells or tissues using the SV Total RNA Isolation Kit (Promega, USA) and reverse transcribed to synthesize first-strand cDNA using the Evo M-MLV qPCR RT Kit (AG, China), following manufacturers’ protocols. qRT-PCR reactions were performed with SYBR premix ExTaq (Takara, Japan) on a LightCycler 480 instrument (Roche Diagnostics, Switzerland), as previously described (40). Expression levels of immune-related genes and viral genes were normalized to scβ-actin gene. Primer sets for the scRNF5, scSTING, scβ-actin, mrvMCP, mrvICP18, and mrvDNA polymerase (mrvDNA-Poly) genes are described in Supplementary Material 1 . Data were analyzed using Q-gene statistics add-in and unpaired sample t-test. Statistical significance was set at p<0.05, with high significance at p<0.01. All data were expressed as mean ± standard deviation (SD).

For Co-IP, FHM cells were seeded in 25 cm² dishes, cultured overnight, and co-transfected with 4 μg of vector. After 24 h, cells were lysed using IP lysis buffer (Beyotime, China) with protease inhibitor cocktail III (Merck Millipore, USA), and whole-cell lysates were centrifuged at 12,000 ×g for 10 minutes at 4°C. Supernatants were collected for immunoprecipitation (IP) using anti-Flag or anti-Myc beads (Alpa Life Bio, China), as previously described [36]. Beads were added to the supernatant, incubated for 4 h at 4°C, washed, and proteins were collected by centrifugation and heated to 100°C for 10 minutes for WB analysis. Protein bands were detected using the High-sig ECL western blotting substrate (Tanon, China) and visualized with the Amersham Image Quant 800 system (Cytiva, USA).

MFF-1 cells were seeded into 96-well plates, cultured overnight to exceeding 80% confluence, and infected with serial 10-fold dilutions of MRV-containing samples, with eight wells per dilution. Eight wells containing 100 μL of virus-free culture medium served as negative controls, and plates were incubated at 27°C for seven days, as previously described (41, 42). The number of positive and negative wells was recorded to calculate the TCID₅₀ using the Spearman-Karber method, as previously described (41, 42).

To investigate the role of RNF5 in mandarin fish, we cloned the full-length cDNA of scRNF5 gene and constructed expression plasmids. Sequence analysis showed that the scRNF5 coding sequence (NCBI accession number: XP_044026571.1) consists of 648 nucleotides. It encodes a 216 amino acids (aas) protein with two transmembrane domains (TM) and a ring-finger domain (RING), weighting 23.2 kDa ( Figure 1A ). 3–D structure predictions showed that the spatial structure of scRNF5 and the spatial arrangement of its domains were similar to its mammalian ortholog ( Figure 1B ). Phylogenetic analysis clustered scRNF5 with RNF5 proteins from other fish, distinguishing it from the other RNF5 family members ( Figure 1C ; Supplementary Material 2 ).

To assess the tissue-specific expression of RNF5 in mandarin fish, we used qRT-PCR to analyze transcript levels of scRNF5 across various tissues, including blood, brain, liver, spleen, fin, gill, intestine, heart, skin, head kidney, and muscle. As shown in Figure 1D , scRNF5 transcripts were detected in all sampled tissues from healthy individuals, with notably higher expression in blood, approximately 81-fold higher than in muscle, highlighting its tissue-specific expression. In MRV-infected cells, scRNF5 expression exhibited a time-dependent increase, with up-regulation levels reaching 18-, 24-, and 60-fold at 24, 36, and 48 h post-infection, respectively ( Figure 1E ), suggested that scRNF5 responded to MRV infection.

MFF-1 cells expressing scRNF5 or a control vector (pCMV-myc) were infected with MRV, and subsequent cells were then harvested for qRT-PCR, WB, and TCID₅₀ analysis. The results showed that overexpression of scRNF5 ( Figure 2A ) reduced the expression level of scIFN-h gene ( Figure 2B ); but the expression levels of the and MRV viral genes (mrvMCP, mrvICP18, mrvDNA-Poly) were significantly higher in cells expressing scRNF5 compared to the control group ( Figures 2C–E ). Furthermore, both the levels of viral titers ( Figure 2F ) and viral protein mrvORF097L ( Figure 2G ) were markedly elevated in the scRNF5 group compared to the control. These results indicated that scRNF5 facilitated MRV replication.

In mammals, STING serves as a key adaptor in innate immune responses triggered by pathogenic DNA or RNA, but its antiviral activity is attenuated by RNF5 (12). Previously, we found that overexpressing scSTING enhances IFN promoter activity in MFF-1 cells (25). To further explored the role of scRNF5 in the IFN signaling pathway, MFF-1 cells were co-transfected with plasmids expressing scRNF5 and/or scSTING, followed by a luciferase reporter assay. The results showed that scSTING, but not scRNF5, significantly activated the IFN-β promoter. Notably, scRNF5 inhibited scSTING-mediated IFN-β-luc activity in a dose-dependent manner when co-expressed with scSTING ( Figures 3A, B ). Furthermore, co-transfection experiments revealed that the expression levels of IFN-I signaling related genes (scIFN-h, scMx, scISG15 genes; Figures 3C–E ) and pro-inflammatory factor (scTNF-α gene; Figure 3F ) in cells co-expressing scSTING and scRNF5 were significantly lower than those in cells co-expressing scSTING and control vector pCMV-myc (p<0.01). These results suggested that scRNF5 might suppress the scSTING-mediated IFN signaling pathway.

To further explored the mechanism of how scRNF5 regulated the scSTING IFN signaling pathway, we conducted a Co-IP assay to analyze the interaction between scSTING and scRNF5. Figure 4A shows a distinct band representing scRNF5 in the Flag-scSTING immunoprecipitated protein complex. Likewise, Figure 4B depicts a distinct band for scSTING in the Myc-scRNF5 immunoprecipitated protein complex. These findings confirmed the interaction between scRNF5 and scSTING. Upon co-transfection of scRNF5 with scSTING, WB analysis showed a significant reduction in scSTING protein levels in the RNF5-expressing group ( Figure 4C ). To further investigate this effect, we employed a gradient of scRNF5 expression and observed a clear inverse relationship; increasing scRNF5 levels correlated with a progressive decrease in scSTING protein levels ( Figure 4D ). Next, we explored the mechanism by which scRNF5 mediated scSTING degradation. Co-transfected of scRNF5 and scSTING, followed by treatment with various drugs including MG132 (specific proteasome inhibitor), 3-MA (autophagy Inhibitor), CQ, and NH₄Cl (lysosomal inhibitors), revealed that MG132 effectively blocked scRNF5-mediated scSTING degradation ( Figure 4E ). Dose-dependent experiments with MG132 further confirmed that it inhibited scRNF5-mediated scSTING degradation in a dose-dependent manner ( Figure 4F ). Based on these results, we hypothesized that scRNF5 mediated scSTING degradation through ubiquitination. Ubiquitination experiments on scSTING in the presence of scRNF5 revealed a significant increase in scSTING ubiquitination levels compared to control samples ( Figure 4G ). These observations suggested that scRNF5 targeted scSTING for degradation via ubiquitination pathway.

To identify the specific ubiquitination site of scSTING by scRNF5, we constructed various truncation mutants of scSTING ( Figure 5A ) and conducted degradation experiments. Initial results showed that scRNF5 degraded both scSTING (1–140) and scSTING(141-417) ( Figure 5B ). However, further segmentation analysis showed that scRNF5 only degraded scSTING(1-180), but not scSTING (181-417) ( Figure 5C ), implying the ubiquitination site is located within the first 180 aas of scSTING. Moreover, ubiquitination experiments on scSTING(1-180) and scSTING(181-417) confirmed significant ubiquitination of scSTING(1-180) by scRNF5 ( Figure 5D ), further indicating that the ubiquitination occurred within the 1 to 180 aas region.

Ubiquitination primarily targets lysine residues in various proteins, although other aas, like cysteine, can also undergo this post-translational modification (25). Mammalian RNF5 suppresses K48-linked ubiquitination at the K150 site, promoting proteasomal degradation (12). To investigated RNF5-mediated ubiquitination of STING involves amino acid residues in mandarin fish, we substituted all C and K residues in scSTING with R, generating a variant named scSTINGCKR. Moreover, we constructed a variant where all K residues in scSTING(1-180) were replaced with R, named scSTING(1-180)KR. Degradation experiments observed that scRNF5 could no longer degrade scSTINGCKR ( Figure 6A ) and scSTING(1-180)KR ( Figure 6B ), suggesting scRNF5-mediated ubiquitination of scSTING specifically targeted lysine residues. As shown in Figure 6C , the 1-180 aa region of scSTING contains six lysine residues. Next, we generated six mutants, each restoring one of the original K residue in the scSTINGCKR, and designed scSTINGCKR20K, scSTINGCKR95K, scSTINGCKR117K, scSTINGCKR122K, scSTINGCKR135K, and scSTINGCKR155K, respectively. We then performed co-transfection with the mutants and scRNF5 was to identify the specific K residue targeted for degradation. The results showed that scRNF5 had no degradation effect on scSTINGCKR20K, scSTINGCKR95K, scSTINGCKR117K, scSTINGCKR122K ( Figures 6D–G ). However, scRNF5 regained its ability to degrade scSTINGCKR when K mutations were restored at positions K135 and K155 ( Figures 6H–I ). Those results suggested that scRNF5 catalyzed the ubiquitination of scSTING specifically at lysine residues K135 and K155.

Furthermore, to compare the differences in scRNF5 targeting of scSTING lysine residues between mammals and other vertebrates, we performed homologous sequence alignment of STING proteins from mammals, birds, reptiles, amphibians and fish. Interestingly, STING from most fish species contains two lysine residues at positions K135 and K155, whereas STING from other vertebrates (mammals, birds, reptiles, and amphibians) does not contain this two lysine ( Figure 7 ), suggesting potential differences in the negative regulation of STING in teleost fish compared to other vertebrates.

To investigate whether the antiviral ability of scSTING was also regulated by scRNF5, MFF-1 cells co-expressing scRNF5 and scSTING were infected with MRV. Viral titer was then assayed from collected supernatants, while cells were harvested for qRT-PCR and WB analysis. The results showed that cells co-expressing scSTING and scRNF5 ( Figures 8A, B ) had increased the expression levels of the scIFN-h gene ( Figure 8C ) and MRV viral genes (mrvMCP, mrvICP18, mrvDNA-Poly) ( Figures 8D–F ). In addition, these cells exhibited higher viral titers (TCID₅₀) and more abundant viral proteins (mrvORF097L) compared to control cells transfected with scSTING alone ( Figures 8G, H ). These results suggested that the significant negative regulation of scRNF5 on scSTING-mediated antiviral innate immunity.