RNF122 targets STING for ubiquitination at residues K95, K117, and K155 to regulate antiviral responses in a teleost fish

Abstract

Ring finger protein 122 (RNF122), an E3 ubiquitin ligase, orchestrates antiviral immune responses in mammals by targeting retinoic acid-inducible gene 1 and melanoma differentiation-associated gene 5 for ubiquitination. However, its functional relevance in teleosts has yet to be clearly defined, particularly regarding the identification of substrate-specific regulatory sites. This study characterized RNF122 from mandarin fish (Siniperca chuatsi), termed scRNF122, and investigated its regulatory impact on stimulator of interferon genes (STING)-mediated antiviral signaling. Results showed that scRNF122 expression was up-regulated in response to mandarin fish ranavirus (MRV) infection, and its overexpression suppressed scSTING-mediated interferon (IFN) production and enhanced MRV replication. Co-immunoprecipitation confirmed a direct interaction between scRNF122 and scSTING. Functional assays demonstrated that scRNF122 facilitated scSTING degradation through the ubiquitin-proteasome pathway, a process impeded by MG132 treatment. Ubiquitination analyses of various scSTING mutants revealed that scRNF122 catalyzed scSTING ubiquitination at K95, K117, and K155 residues. Moreover, scRNF122 significantly impaired scSTING-dependent antiviral responses by engaging negative regulatory elements within the signaling cascade. Overall, scRNF122 was identified as a negative modulator of STING-mediated IFN signaling in mandarin fish, diminishing STING-dependent antiviral activity and promoting its degradation via the ubiquitin-proteasome pathway at lysine residues K95, K117, and K155. These findings provide mechanistic insight into the post-translational control of STING in teleosts and establish a foundation for future investigations into antiviral immune regulation.

INTRODUCTION

The innate immune system constitutes the first line of defense against viral pathogens, with the stimulator of interferon genes (STING) pathway serving as a primary sensor of viral DNA. STING, a transmembrane adapter protein, plays an essential role in detecting pathogenic cytoplasmic DNA (Ishikawa & Barber, 2008; Zhong et al., 2008) and initiating downstream signaling cascades that culminate in the production of type I interferon (IFN-I) and proinflammatory cytokines, thereby triggering a robust antiviral immune response (Ishikawa et al., 2009; Phelan et al., 2020). This DNA-sensing axis is a crucial component of host recognition and response to viral infections, and its activity is tightly regulated through various post-translational modifications, including phosphorylation and ubiquitination (Hopfner & Hornung, 2020). Among these modifications, ubiquitination has emerged as a critical modulator of STING function, exerting context-dependent effects that either enhance or suppress signaling output (Bhoj & Chen, 2009; Pan et al., 2023).

Recent studies have highlighted RNF122, an E3 ubiquitin ligase, as a pivotal negative regulator of antiviral responses in mammals. RNF122 mediates proteasomal degradation of retinoic acid-inducible gene 1 (RIG-I) by targeting its CARD, thereby dampening RIG-I-dependent signaling (Wang et al., 2016). Additionally, during porcine reproductive and respiratory syndrome viral infection, non-canonical viral proteins such as Nsp1a, Nsp7, and Nsp9 induce RNF122 expression, leading to the ubiquitination and degradation of melanoma differentiation-associated gene 5 (MDA-5), thereby facilitating viral replication (Sun et al., 2022). Despite these insights, the role of RNF122 in teleost fish remains incompletely characterized, particularly in relation to substrate recognition and site-specific regulatory activity (Sun et al., 2022).

The mandarin fish (Siniperca chuatsi) is an economically important species in Chinese aquaculture (Yu et al., 2023), yet its intensive cultivation has led to increased susceptibility to infectious outbreaks. Among these, mandarin fish ranavirus (MRV), a double-stranded DNA virus belonging to the Ranavirus genus of the Iridoviridae family, presents a major threat to aquaculture sustainability (Dong et al., 2017). Elucidating the mechanisms by which innate immunity regulates viral control in this species is crucial for advancing fish health management and antiviral intervention strategies. This study investigated the immunoregulatory function of S. chuatsi RNF122 (scRNF122) in modulating STING-mediated antiviral immune responses and identified the specific lysine residues on STING targeted for ubiquitination. The results of this study offer valuable insights into the regulatory mechanisms underlying STING-mediated signaling in teleost fish.

MATERIALS AND METHODS

Animals, cells, and viruses

Mandarin fish (average weight 50 g) were obtained from a farm located in Guangdong Province, China. Before experimentation, the fish were acclimatized for two weeks in a recirculating freshwater system maintained at 27°C. All animal procedures were performed in accordance with the regulations for animal experimentation in Guangdong Province, China, and permitted by the Ethics Committee of Sun Yat-sen University (approval no. 2023121901). Mandarin fish fry (MFF-1) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) at 27°C in a humidified atmosphere containing 5% CO₂ (Dong et al., 2008). Fathead minnow (FHM) cells (ATCC CCL-42) were maintained in M199 medium supplemented with 10% FBS at 27°C (Li et al., 2024). Human normal liver cells (L02) were maintained in DMEM supplemented with 10% FBS at 37°C. The MRV strain NH-1609 (MG941005) was isolated from mandarin fish in Nanhai, Guangdong, China, and stored in our laboratory (Li et al., 2024).

Reagents and antibodies

Proteasome inhibitor MG132, cycloheximide (CHX), 3-methyladenine (3-MA), and chloroquine (CQ) were purchased from MedChemExpress (USA). Iodoacetamide (IAA) was obtained from Cell Signaling Technology (CST, USA). Mouse monoclonal antibodies against C-Myc and Flag were sourced from Sigma-Aldrich (1:5 000, USA). Anti-HA monoclonal antibody was obtained from CST (1:1 000, USA) and anti-GAPDH antibody was procured from Abways (1:3 000, China). Rabbit polyclonal anti-mrvORF097L antibody (1:5 000) was generated in our laboratory (Pan et al., 2024). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) secondary antibodies were obtained from Promega (1:5 000, USA).

Bioinformatic analyses

Protein domains of scRNF122 were predicted using the SMART database (http://smart.embl-heidelberg.de/) in Genomic Mode (Letunic et al., 2021). Protein sequences were retrieved from GenBank (Benson et al., 2013) and RefSeq (O'Leary et al., 2016) for phylogenetic analysis. Sequences were aligned using ClustalW in MEGA v.11.0.13 (Tamura et al., 2021). The phylogenetic tree was constructed using the neighbor-joining method with 1 000 bootstrap replications under the Jones-Taylor-Thornton (JTT) model and complete deletion settings. Tree visualization was performed using iTOL (https://itol.embl.de/) (Letunic & Bork, 2024). Illustrative elements were prepared using the Generic Diagramming Platform (Jiang et al., 2025). Protein amino acid sequences retrieved from GenBank and RefSeq were aligned using Dynamic Alignment with default parameters in DNAMAN v.9.0.

Plasmid construction and cell transfection

Total RNA was extracted using an SV Total RNA Isolation Kit (Promega, USA) and reverse transcribed into first-strand cDNA using an Evo M-MLV qPCR RT Kit (Accurate Biology, China), as described in previous research (Qin et al., 2020). The scRNF122 and scSTING gene fragments were cloned and inserted into pCMV-Myc-N and pCMV-Flag-N vectors (Takara, Japan), respectively. Various scSTING gene segments, including scSTING(1–94), scSTING(1–121), scSTING(1–116), scSTING(1–135), scSTING(1–140), scSTING(1–180), scSTING(181–417), scSTINGCKR(1–161)155K, scSTINGCKR(1–141)135K, scSTINGCKR(1–128)122K, scSTINGCKR(1–123)117K, and scSTINGCKR(1–101)95K, were processed. In addition, multiple scSTING constructs, including Flag-scSTING mutants (e.g., scSTING(1–180)KR, scSTINGCKR, scSTINGCKRK20, scSTINGK20R, scSTINGCKRK95, scSTINGK95R scSTINGCKRK117, scSTINGK117R, scSTINGCKRK122, scSTINGCKRK135, scSTINGCKRK155, scSTINGK155R, and scSTINGK95/117/155R), were produced by Beijing Tsingke Biotechnology (China). The primers used for gene cloning, which incorporated restriction enzyme sites, are detailed in Supplementary Table S1. Transfection of the plasmids into FHM cells was carried out using Fugene HD (Promega, USA), while MFF-1 cells were transfected using Transfect EZ 3000 Plus (eLGbio, China), following the manufacturer’s protocols.

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)

RT-qPCR was performed using SYBR Premix ExTaq (Takara, Japan) on a LightCycler 480 system (Roche Diagnostics, Switzerland), following previously established protocols (Qin et al., 2020). Expression levels of immune-related and viral genes were normalized to scβ-actin. Primer sequences for scRNF122, scSTING, scβ-actin, mrvMCP, mrvICP18, and mrvDNA polymerase (mrvDNA-Poly) genes are provided in Supplementary Table S2.

Tissue expression profiling and dual-luciferase reporter assays

To determine the tissue-specific distribution of scRNF122 in healthy fish, total RNA was extracted from blood, brain, gills, fins, spleen, intestine, head kidney, middle kidney, hind kidney, fat, heart, liver, and muscle, as previously described (Qin et al., 2020). RT-qPCR was employed to assess transcript levels across tissues using the primer sets listed in Supplementary Table S2. For promoter activity analysis, luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, USA), following established procedures (Qin et al., 2020). All experiments were conducted in triplicate, with data representing the mean of at least three independent experiments.

Co-immunoprecipitation (Co-IP) and western blot (WB) analysis

The Co-IP and WB procedures were carried out as previously reported (Qin et al., 2020). Protein bands were visualized using High-sig ECL western blotting substrate (Tanon, China) and imaged with the Amersham ImageQuant 800 system (Cytiva, USA).

Viral titer determination

Virus titers in MFF-1 cells were quantified according to previously described protocols (Zeng et al., 2021). In brief, cells were seeded into 96-well plates and incubated until reaching a confluence of over 80%. Serial 10-fold dilutions of MRV-containing samples were prepared and inoculated into eight replicate wells per dilution. Negative control wells were inoculated with 100 μL of virus-free culture medium. Plates were incubated at 27°C for seven days, after which the number of positive and negative wells was recorded. TCID₅₀ values were calculated using the Spearman-Karber method (Reed & Muench, 1938).

Statistical analysis

Statistical analyses were performed using SPSS v.20. One-way analysis of variance (ANOVA) was applied to assess differences between groups. Data are presented as mean±standard deviation (SD), derived from three independent experiments performed in triplicate. Statistical significance was defined as ^*: P<0.05 and ^**: P<0.01 compared to the controls.

RESULTS

Characterization of scRNF122 and its promotion of MRV replication

Sequence alignment of mandarin fish transcriptomic data revealed a transcript (XM_044191507.1) with high homology to RNF122. The corresponding gene was cloned, resulting in the acquisition of a 468 bp full-length cDNA, designated scRNF122. SMART domain analysis and amino acid sequence examination indicated that scRNF122 encodes a 17.6 kDa protein containing a transmembrane region (TM) and canonical RING-finger domain (RING) (Figure 1A). Phylogenetic analysis positioned scRNF122 within the same clade as RNF122 proteins from other fish species, distinct from other RNF122 family members (Figure 1B; Supplementary Table S3). Expression profiling of scRNF122 across multiple tissues in healthy mandarin fish showed that scRNF122 was broadly expressed, with the highest expression observed in spleen—a crucial immune tissue in fish—approximately 27-fold higher than that in muscle (Figure 1C), suggesting potential involvement in immune response.

Figure 1.

Characterization of scRNF122 and its role in promoting viral replication

A: Predicted domain structure of scRNF122. B: Phylogenetic analysis of RNF122 sequences from representative vertebrate species (GenBank accession numbers are provided in Supplementary Table S3). C: Tissue distribution of scRNF122 protein expression in healthy mandarin fish. Total RNA was extracted from blood, brain, liver, spleen, fin, gill, intestine, heart, skin, head kidney, and muscle samples obtained from three healthy mandarin fish, with scRNF122 expression quantified by RT-qPCR. D: Induction of scRNF122 in MFF-1 cells following MRV infection (multiplicity of infection (MOI) of 0.1), with scRNF122 mRNA expression determined by RT-qPCR and normalized to β-actin. Cells were transfected with Myc-scRNF122 or pCMV-Myc for 24 h, then infected with MRV and harvested at indicated time points. E–I: RT-qPCR quantification of scRNF122, scIFN-h, mrvMCP, mrvICP18, and mrvDNA-Poly expression in MRV-infected cells at indicated times. Expression values were normalized to the lowest-expressing group, set to 1. J: Viral titers determined by endpoint dilution assay in 96-well culture plates. K: Detection of mrvORF097L protein levels by WB analysis. L: Densitometric quantification of mrvORF097L normalized to GAPDH. Vertical bars represent ±SD (n=3). ^**: P<0.01.

To investigate whether scRNF122 participates in the host response to viral infection, its transcriptional dynamics were monitored in MFF-1 cells following MRV challenge. Results showed that scRNF122 expression increased in a time-dependent manner, exhibiting 47-, 141-, and 158-fold up-regulation at 24, 36, and 48 h post-infection, respectively (Figure 1D), indicating potential involvement in viral pathogenesis. Functional analysis was performed by overexpressing scRNF122 in MFF-1 cells, followed by MRV infection. Cells transfected with either scRNF122 or the control vector (pCMV-Myc) and subsequently infected with MRV were collected for RT-qPCR, WB, and TCID₅₀ assays. Overexpression of scRNF122 (Figure 1E) led to a marked decrease in scIFN-h expression (Figure 1F), whereas transcription of MRV genes mrvMCP, mrvICP18, and mrvDNA-Poly was significantly elevated compared to controls (Figure 1G–I). Correspondingly, viral titers (Figure 1J) and levels of the viral protein mrvORF097L (Figure 1K, L) were markedly increased in the scRNF122 group compared to controls. These observations indicate that scRNF122 inhibits the production of IFN and enhances MRV replication.

scRNF122 targets the 141–417 amino acid region of STING to suppress IFN signaling

To explore the regulatory role of scRNF122 in STING-mediated IFN signaling, dual-luciferase reporter assays were conducted following co-transfection of cells with plasmids encoding scRNF122 and scSTING. Results showed that when both proteins were co-expressed, scRNF122 exerted a dose-dependent inhibitory effect on scSTING-induced IFN-β-luciferase activity (Figure 2A, B). Furthermore, expression levels of IFN-I-related genes (scIFN-h, scMx, and scISG15; Figure 2C–E) and the pro-inflammatory cytokine scTNF-α (Figure 2F) were significantly decreased in cells co-expressing scSTING and scRNF122 compared to those co-expressing scSTING and the pCMV-Myc control vector (P<0.01). These findings suggest that scRNF122 acts as a negative regulator of scSTING-mediated IFN signaling.

Figure 2.

scRNF122 inhibits scSTING-induced IFN signaling and physically interacts with scSTING

A: Relative IFN-β-luc activity was measured in cells co-transfected with Flag-scSTING and Myc-scRNF122. Cells transfected with pCMV-Myc alone served as the negative control. B: MFF-1 cells were co-transfected with pRL-TK and IFN-β-luc reporter plasmids, along with scRNF122 and scSTING expression plasmids in 24-well plates. Luciferase activity was assessed 36 h post-transfection. Vertical bars represent ±SD (n=3). ^**: P<0.01. C–F: mRNA expression levels of scIFN-h (C), scMx (D), scISG15 (E), and scTNF-α (F) were quantified by RT-qPCR 24 h after co-transfection with scSTING and scRNF122 or vector. Data represent three independent experiments (n=3). ^**: P<0.01. G, H: FHM cells were transfected with corresponding plasmids or empty vectors, followed by Co-IP and WB analyses to examine the interaction between scRNF122 and scSTING. IB: Immunoblotting; IP: Immunoprecipitation. I: FHM cells were co-transfected with pCMV-Myc-scRNF122 or empty vector, pCMV-Flag-scSTING(1–140), and pCMV-Flag-scSTING(141–417) plasmids. Co-IP and WB analyses were performed to map interaction regions.

To determine the region of scSTING targeted by scRNF122, Co-IP assays were performed. A prominent band corresponding to scRNF122 was detected in the Flag-scSTING immunoprecipitated protein complex (Figure 2G). Similarly, a clear band for scSTING was observed in the Myc-scRNF122 immunoprecipitated complex (Figure 2H), confirming their interaction. Segmental analysis of scSTING further revealed that the interaction was mediated through the 141–417 amino acid region (Figure 2I). These results indicate that scRNF122 exerts its inhibitory effect on IFN signaling by binding to the 141–417 amino acid region of scSTING.

scRNF122 targets the 94–180 amino acid region of STING for ubiquitination and subsequent degradation

To elucidate the role of scRNF122 in modulating scSTING stability, WB analyses were performed to assess scSTING levels and investigate its ubiquitination and degradation. Results showed that co-transfection of scSTING and scRNF122 led to a significant reduction in scSTING levels in cells expressing scRNF122 (Figure 3A). Moreover, this effect was dose-dependent, with increasing levels of scRNF122 expression leading to progressive depletion of scSTING (Figure 3B). To investigate the mechanism underlying scRNF122-induced scSTING degradation, the co-transfected cells were treated with various inhibitors, including the proteasome inhibitor MG132, autophagy inhibitor 3-MA, and lysosomal inhibitors CQ and NH₄Cl. Among these, MG132 effectively prevented scRNF122-mediated scSTING degradation (Figure 3C). Dose-response experiments confirmed the dose-dependent inhibitory effects of MG132 on scSTING degradation (Figure 3D). These observations suggested that scRNF122 promoted scSTING degradation through ubiquitination. Consistent with this hypothesis, ubiquitination assays revealed significantly higher levels of scSTING ubiquitination in the presence of scRNF122 compared to controls (Figure 3E). Taken together, these results indicate that scRNF122 targets scSTING for degradation via the ubiquitin-proteasome pathway.

Figure 3.

scRNF122 mediates scSTING degradation via ubiquitination targeting the 94–180 amino acid region

A: WB analyses were used for detection. FHM cells were transfected with pCMV-Myc-scRNF122 or an empty vector alongside pCMV-Flag-scSTING plasmids. B: FHM cells were seeded in 6-well plates, incubated overnight, and co-transfected with 2 μg Flag-scSTING and Myc-scRNF122 (0.5, 1, and 2 μg), After 24 h, lysates were immunoblotted with anti-Flag, anti-Myc, and anti-GAPDH antibodies. C: Effects of MG132, 3-MA, CQ, and NH₄Cl on scRNF122-induced scSTING degradation were assessed by treating FHM cells in 6-well plates with 2 μg of Flag-scSTING and 2 μg of Myc-scRNF122. At 24 h post-transfection (hpt), cells were treated with DMSO or respective inhibitors (MG132: 20 μmol/L, 3-MA: 5 μmol/L, CQ: 10 μmol/L, NH₄Cl: 20 μmol/L). After 12 h, lysates were immunoblotted using anti-Flag, anti-Myc, and anti-GAPDH antibodies. D: FHM cells were transfected and treated with increasing MG132 concentrations (1, 10, and 20 μmol/L), and a negative control (MG132). Lysates were immunoblotted as before. E: Ubiquitination of scSTING was assessed in FHM cells co-expressing Flag-scSTING, Ha-ubiquitin, and either Myc-scRNF122 (lane 2) or empty vector (lane 1). Flag-scSTING was immunoprecipitated with anti-Flag, and poly-ubiquitin chains were detected with anti-Ha. F: Positions of lysine (K) residues in truncated STING variants. G, H: FHM cells were seeded overnight in 6-well plates and co-transfected with 2 μg of either Flag-scSTING(1–180) or Flag-scSTING(181–417), along with Myc-scRNF122 at concentrations of 0.5, 1, or 2 μg. After 24 h, lysates were immunoblotted using anti-Flag, anti-Myc, and anti-GAPDH antibodies. I: Ubiquitination of scSTING fragments was assessed by co-transfecting FHM cells with Myc-scRNF122, Ha-ubiquitin, and either Flag-scSTING(1–180) or Flag-scSTING(181–417). Flag-scSTING was immunoprecipitated using anti-Flag, and poly-ubiquitin chains were detected with anti-Ha. J–N: Similarly, FHM cells were seeded and co-transfected as in C, but with Flag-scSTING(1–140), Flag-scSTING(1–134), Flag-scSTING(1–121), Flag-scSTING(1–116), or Flag-scSTING(1–94). Lysates were analyzed by immunoblotting as described above.

To pinpoint this specific ubiquitination site, a series of truncated scSTING variants were generated (Figure 3F) and subjected to degradation assays. Results showed that scRNF122 selectively targeted the N-terminal segment of scSTING (1–180 amino acids) for degradation, with the C-terminal region (181–417 amino acids) remaining stable (Figure 3G, H). Further refinement using N-terminal truncations revealed that constructs spanning residues 1–140, 1–134, 1–121, and 1–116 were all susceptible to degradation, while the variant containing residues 1–94 was unaffected (Figure 3J–N), implying that the ubiquitination sites reside within the 94–180 amino acid region of scSTING. Supporting this conclusion, scRNF122 induced strong ubiquitination of the scSTING(1–180) fragment but not of the scSTING(181–417) fragment (Figure 3I), confirming that the ubiquitination sites are confined within the 94–180 amino acid segment.

scRNF122 mediates scSTING ubiquitination at three specific lysine residues

Ubiquitination primarily targets lysine residues, although cysteine residues may also be modified (Hershko & Ciechanover, 1998). To determine whether scRNF122 specifically targets lysine residues for scSTING ubiquitination, a mutant construct, scSTINGCKR, was generated by replacing all cysteine (C) and lysine (K) residues with arginine (R) (Figure 4A). An additional mutant, scSTING(1–180)KR, with all K residues in the N-terminal 1–180 amino acids substituted with R, was also constructed. Degradation assays demonstrated that scRNF122 failed to degrade either scSTINGCKR (Figure 4B) or scSTING(1–180)KR (Figure 4C), suggesting that scRNF122-mediated ubiquitination of scSTING is lysine-specific. The 1–180 amino acid region of scSTING contains six K residues. To identify which are targeted, a series of truncated rescue constructs was generated based on scSTINGCKR(1–180), each restoring a single original lysine, including scSTINGCKR(1–101)95K, scSTINGCKR(1–123)117K, scSTINGCKR(1–128)122K, scSTINGCKR(1–141)135K, and scSTINGCKR(1–161)155K. Co-transfection experiments revealed that scRNF122 selectively degraded the 95K, 117K, and 155K rescue mutants (Figure 4D, G, H), whereas constructs restoring 122K and 135K were resistant to degradation (Figure 4E, F), identifying K95, K117, and K155 as the primary ubiquitination targets. To validate these findings in full-length scSTING, six mutants were generated from STINGCKR, including scSTINGCKR20K, scSTINGCKR95K, scSTINGCKR117K, scSTINGCKR122K, scSTINGCKR135K, and scSTINGCKR155K (Figure 4I). Co-transfection experiments showed that scRNF122 failed to degrade scSTINGCKR20K, scSTINGCKR122K, and scSTINGCKR135K (Figure 4J, M, N), but successfully induced degradation of K95, K117, and K155 (Figure 4K, L, O). In addition, scRNF122 also degraded human STING in a dose-dependent manner in L02 cells (Figure 4P). These results indicate that scRNF122 catalyzes the ubiquitination of scSTING specifically at lysine residues K95, K117, and K155.

Figure 4.

scRNF122 targets scSTING for ubiquitination at K95, K117, and K155

A: Schematic of scSTING mutants used in this study. B, C: FHM cells were co-transfected with 2 μg of Flag-scSTINGCKR/Flag-scSTING(1–180)KR and Myc-scRNF122 (0.5, 1 and 2 μg) for 24 h. Lysates were then subjected to immunoblotting with anti-Flag, anti-Myc, and anti-GAPDH Abs. D–H: FHM cells were co-transfected with 2 μg of various Flag-tagged scSTING mutants, including scSTINGCKR(1–161)155K, scSTINGCKR(1–141)135K, scSTINGCKR(1–128)122K, scSTINGCKR(1–123)117K, and scSTINGCKR(1–101)95K, along with Myc-scRNF122 (0.5, 1, and 2 μg) for 24 h. Lysates were then immunoblotted with anti-Flag, anti-Myc, and anti-GAPDH Abs. I: Additional schematic of scSTING mutants employed in this study. J–O: FHM cells were seeded and co-transfected as in B, but with specific scSTINGCKR mutants: scSTINGCKR20K, scSTINGCKR95K, scSTINGCKR117K, scSTINGCKR122K, scSTINGCKR135K and scSTINGCKR155K. Lysates were processed for immunoblotting. P: L02 cells were seeded and co-transfected as in B, but with hsSTING. Lysates were analyzed as described above.

To further dissect the relative contributions of these lysine residues, individual lysine-to-arginine mutations were introduced to generate scSTINGK95R, scSTINGK117R, and scSTINGK155R, along with a triple mutant scSTINGK95/117/155R (Figure 5A). Co-transfection of these mutants with scRNF122 revealed that the single mutants remained susceptible to degradation, whereas the triple mutant was resistant (Figure 5B). Ubiquitination assays confirmed that scSTING ubiquitination mediated by scRNF122 significantly decreased with increasing STING site mutations (Figure 5C). Luciferase assays further showed that IFN activation was partially restored by single-site mutations, with the K155 mutant exhibiting the strongest restoration (Figure 5D). These findings indicate that scRNF122 is capable of ubiquitinating all three lysine residues—K95, K117, and K155. However, mutation at K155 results in a markedly greater restoration of IFN activity than mutations at K95 or K117, suggesting that K155 serves as the dominant site mediating scRNF122-dependent suppression of STING signaling.

Figure 5.

scRNF122 ubiquitinates specific lysine residues (K95, K117, and K155) of scSTING

A: Schematic representation of scSTING mutants used in this study. B: FHM cells were co-transfected with 2 μg of Flag-scSTING mutants (K95R, K117R, K155R, and K95/117/155R) and increasing amounts of Myc-tagged scRNF122 (0.5, 1, and 2 μg) for 24 h. Cell lysates were then subjected to immunoblotting with anti-Flag, anti-Myc, and anti-GAPDH antibodies. C: Ubiquitination of scSTING mutants (K95R, K117R, K155R, and K95/117/155R) was assessed. FHM cells were co-expressed with Myc-scRNF122, Ha-ubiquitin, and either Flag-scSTING, Flag-scSTINGK95R, Flag-scSTINGK117R, Flag-scSTINGK155R, or Flag-scSTINGK95/117/155R. Flag-scSTING was immunoprecipitated using anti-Flag, and poly-ubiquitin chains were detected with anti-Ha. D: MFF-1 cells were co-transfected in 24-well plates with reporter plasmids pRL-TK and IFN-β-luc, along with plasmids encoding scRNF122 or pCMV-Myc and scSTING mutants. Luciferase activity was measured 36 h post-transfection. E: Protein amino acid alignment revealed conserved amino acid sequences and distribution of lysine sites in STING across different species. Accession numbers for these sequences are provided in Supplementary Table S3. Arrows indicate lysine residues at positions 95, 117, and 155 in scSTING. Asterisk (^*) represents fish belonging to the order Perciformes.

To explore the differences in scRNF122-mediated K residue targeting scSTING between mammals and other vertebrates, homologous sequences of STING were compared across mammals, birds, reptiles, amphibians, and fish. Notably, lysine 155 (K155) in fish STING exhibited high homology with the highly conserved K150 in other vertebrates (Figure 5E), suggesting functional conservation of this site in negative regulation. In contrast, lysine residues at 95 and 117 were predominantly conserved within Perciformes, indicating that distinct regulatory features may exist among teleost lineages relative to other vertebrates.

scRNF122 negatively regulates scSTING-mediated antiviral activity

To assess the impact of scRNF122 on scSTING-mediated antiviral defense, MFF-1 cells co-expressing both proteins were subjected to MRV infection. Viral loads in the collected supernatants were quantified, and cellular samples were analyzed by RT-qPCR and WB. Co-expression of scSTING and scRNF122 (Figure 6A, B) resulted in increased transcription of the scIFN-h gene (Figure 6C), alongside elevated expression of MRV genes mrvMCP, mrvICP18, and mrvDNA-Poly (Figure 6D–F). In addition, viral titers (TCID₅₀) and levels of viral protein mrvORF097L were significantly higher in the co-expression group compared to cells expressing scSTING alone (Figure 6G, H). Collectively, these results demonstrate that scRNF122 attenuates scSTING-dependent antiviral signaling.

Figure 6.

scRNF122 inhibits scSTING-mediated antiviral activity

A–F: MFF-1 cells were infected with MRV and harvested at the indicated time points for RT-qPCR, WB, and TCID₅₀ analyses. Expression levels of scRNF122 (A), scSTING (B), scIFN-h (C), mrvMCP (D), mrvICP18 (E), and mrvDNA-Poly (F) in MRV-infected cells at indicated times. For RT-qPCR, the baseline was set as the group with the lowest relative expression among all groups, assigned a value of 1. Each group represented different time points after viral infection. G: Virus titers were determined using the endpoint dilution method. H: WB analysis was performed to assess levels of the viral protein mrvORF097L, with densitometric quantification normalized to GAPDH. Left two lanes represent samples collected at 24 hpi, while right two lanes indicate samples collected 48 hpi. Vertical bars represent ±SD (n=3). ^**: P<0.01.

DISCUSSION

This study demonstrated that RNF122 exerted an inhibitory effect on the STING-mediated IFN signaling pathway in mandarin fish, impairing STING-driven antiviral responses by facilitating its degradation through the ubiquitin-proteasome pathway at lysine residues K95, K117, and K155 (Figure 7). These findings elucidate a previously uncharacterized regulatory mechanism of STING activity in teleosts and provide a molecular basis for modulating antiviral immunity in aquatic species, offering potential strategies for the development of disease-resistant strains and immunomodulatory therapeutics.

Figure 7.

Schematic representation of RNF122-mediated negative regulation of STING signaling in mandarin fish

scRNF122 interacted with scSTING and catalyzed ubiquitination at lysine residues K95, K117, and K155. This post-translational modification targeted scSTING for proteasomal degradation, thereby preventing IFN signaling. Through targeted degradation of scSTING, scRNF122 impaired the host antiviral response and promoted viral replication.

Although IFNs are essential components of the innate immune system, their excessive production can provoke pathological inflammation, leading to severe outcomes. Therefore, tight regulation of IFN signaling is vital for preserving immune homeostasis (Jefferies, 2019). TRIM56 promotes K63-linked ubiquitination at the K150 residue of STING, facilitating the activation of the STING/IFN signaling pathway (Tsuchida et al., 2010), while RNF5 inhibits K48-linked ubiquitination at the K150 residue, directing STING toward proteasomal degradation (Zhong et al., 2009). In this study, RNF122 was shown to negatively regulate STING activity in mandarin fish by enhancing its ubiquitination and degradation.

The contribution of RNF family members to antiviral immunity has gained increasing recognition. For instance, RNF144A is up-regulated following HSV-1 infection, which amplifies innate immune reactions triggered by DNA viruses (Yang et al., 2023). Conversely, LjRNF114, induced by infection with red-spotted grouper nervous necrosis virus, attenuates antiviral signaling through the RLR pathways, facilitating immune evasion and viral infection (Xiang et al., 2021). In this context, the present findings demonstrated that MRV infection up-regulated RNF122 expression in mandarin fish cells. Notably, RNF122 expression was also enriched in immune-relevant tissues, including spleen and blood, supporting its pivotal role in modulating the innate immune response during viral challenge.

E3 ubiquitin ligases serve as critical modulators of the antiviral innate immune response by orchestrating the post-translational regulation of IFN-I production (Schneider et al., 2014). Among these, RNF114 suppresses IFN-I induction by interacting with MAVS and impairing antiviral responses during vesicular stomatitis virus (VSV) infection, thereby facilitating viral replication (Han et al., 2022). In contrast, RNF123 enhances IFN-I expression in duck Tembusu viral infection by facilitating TLR3/IRF7 signaling through the degradation of SOCS1, ultimately restricting viral replication (Huang et al., 2023). RNF122 has also emerged as a negative regulator of the RIG-I pathway, targeting the CARD of RIG-I for ubiquitin-mediated degradation (Wang et al., 2016). In pigs, RNF122 promotes MDA-5 ubiquitination and degradation to support porcine reproductive and respiratory syndrome virus replication (Sun et al., 2022), while in carp, RNF122-like protein facilitates spring viremia of carp virus replication by triggering STING degradation via ubiquitination (Liu et al., 2024). Consistent with these roles, our results demonstrated that RNF122 inhibits STING-induced IFN-I production and promotes MRV replication in mandarin fish.

Despite the central role of E3 ligases in shaping antiviral immunity, the identification of precise ubiquitination sites—particularly in non-mammalian vertebrates—remains limited. Prior studies have shown that the RING finger domain of RNF122 mediates ubiquitin transfer to lysine residues K115 and K146 within the CARD of RIG-I (Wang et al., 2016). In the current work, RNF122 was shown to target STING for ubiquitination at lysine residues K95, K117, and K155 in mandarin fish. Notably, RNF122 also induced the degradation of human STING in a dose-dependent manner, indicating a conserved regulatory function across vertebrates. Sequence comparison revealed that K155 in teleost STING shared homology with mammalian K150, while K95 and K117 were only found in Perciformes. This lineage-specific pattern of lysine conservation suggests that RNF122 may modulate STING activity through distinct mechanisms in different fish clades. Such targeted regulation of inflammatory signaling could contribute to enhanced immune tolerance and potentially support increased healthspan in certain teleost lineages (Wang et al., 2024).

While our study offers significant insights into the role of RNF122 in regulating STING-mediated immune responses, several limitations need to be recognized. Firstly, our findings were primarily based on in vitro experiments, and their physiological relevance in vivo remains to be established. Future studies should prioritize in vivo validation using appropriate animal models to assess the functional consequences of RNF122 modulation in more complex biological systems. Targeted gene knockout experiments, such as RNF122 knockout via CRISPR/Cas9, should also be conducted to further clarify the impact of RNF122 deficiency on STING signaling and antiviral immunity. Additionally, investigating the potential of RNF122 as a therapeutic target in aquaculture presents a promising avenue. Strategies such as the development of RNF122-specific inhibitors or the generation of RNF122-deficient fish lines may offer valuable approaches to enhance viral resistance in commercially important teleost species. Given the apparent contribution of RNF122 to dampening inflammatory signaling, such interventions may also support improved immune resilience and healthspan in farmed fish (Wang et al., 2024). These future directions will be essential for evaluating the translational potential of RNF122-targeted manipulation for controlling viral infections in aquaculture settings.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

Funding Statement

This work was supported by the National Key Research and Development Program of China (2022YFE0203900, 2024YFD2401101), China Agriculture Research System (CARS-46), National Natural Science Foundation of China (32473201), and Guangdong S&T Program (2022B1111030001, 2024B1212040007)