HEK-293T (ATCC CRL-11268), SK6 (Wuhan University), and HeLa (ATCC CCL-2) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gen-View, Jacksonville, FL). The primary porcine alveolar macrophages (PAMs) were isolated from 20- to 30-day-old piglets and cultured in RPMI 1640 medium (Gibco, Waltham, MA, USA) supplemented with 10% FBS. All cells were maintained at 37°C, containing 5% CO₂. ASFV strain SXH1 of genotype II was isolated from clinical samples using PAM cells as previously described (28). The enhanced green fluorescent protein-tagged vesicular stomatitis virus (eGFP-VSV) and Sendai virus (SeV) were amplified as previously described and stored at -80°C (29).

The full-length MGF505–6R gene was amplified based on the ASFV strain Wuhan 2019–1 (GenBank accession No. MN393476) and tagged with 6His through cloning into the pTRIP-CMV vector. The truncations of MGF505–6R-N (1–260aa), MGF505–6R-C (261–526aa), STING-TM (1–154aa), STING-NT (1–180aa), STINGΔCTT (1–340aa), and STINGΔTM (154–379aa) were subcloned into the pTRIP-CMV vector with a Flag tag. The plasmids for human Flag-cGAS, Flag-STING, HA-STING, Flag-TBK1, Flag-IKKϵ, and Flag-IRF3 were cloned into the pTRIP-CMV or pcDNA3.1-HA vector. Flag-MAVS and Flag-TRIF were kindly provided by Meilin Jin (Hua Zhong Agricultural University). Porcine cGAS and STING sequences were cloned into the pTRIP-CMV vector to yield Flag-cGAS and Flag-STING expression plasmids. pRL-TK and pGL3-IFN-β-Luc plasmids were used for dual luciferase assays (29). The HA-tagged ubiquitin and ubiquitin mutants were cloned into the pcDNA3.1-HA vector using overlap PCR or mutagenesis kits (Vazyme, China).

Mouse monoclonal antibodies against His-tag (66005–1-Ig), HA-tag (66006–2-Ig), and GAPDH (60004–1-Ig), as well as rabbit polyclonal antibodies against Flag-tag (20543–1-AP), cGAS (26416–1-AP), STING (19851–1-AP), TBK1 (28397–1-AP), IRF3 (11312–1-AP), Phospho-IRF3 (29528–1-AP), ATG7 (10088–2-AP), and LC3B (18725–1-AP) were procured from Proteintech (Rosemont, IL, USA). Rabbit His-tag (PM032) polyclonal antibodies, mouse Flag-tag monoclonal antibody (M185–3L), goat anti-mouse (72–8042-M001), and goat anti-rabbit polyclonal antibodies (72–8067-M001) were purchased from Medical and Biological Laboratories (Nagoya, Japan). A human anti-p54 antibody was prepared as previously described (28). The jetPRIME for transfection was purchased from Polyplus-Transfection (Illkirch, France). Alexa Fluor 555-labeled goat anti-mouse antibody (A-21424), Alexa Fluor 488-labeled goat anti-rabbit antibody (A32731TR), and 4’,6-diamidino-2-phenylindole (DAPI, 62247) were purchased from Invitrogen (Carlsbad, CA, USA). Caspase inhibitor Z-VAD-FMK (HY-16658B) and autophagosome inhibitor chloroquine (CQ, HY-17589A) were procured from MedChemExpress (NJ, USA). Hieff^® qPCR SYBR Green Master Mix (Low Rox Plus) (11202ES03) and Hifair^®V one-step RT-gDNA digestion SuperMix (11142ES10) for qPCR were purchased from YEASEN (Shanghai, China). The proteasome inhibitor MG132, dimethyl sulfoxide (DMSO), firefly luciferase reporter gene assay kit, poly-L-lysine, protease inhibitor cocktail, and protein A+G agarose gel were purchased from Beyotime Biotechnology (Shanghai, China). The nuclear and cytoplasmic extraction kit was acquired from Thermo Fisher Scientific (MA, USA). Cycloheximide (CHX) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

To assess the effects of MGF505–6R overexpression on IFN-β promoter activity. HEK-293T and SK6 cells were co-transfected with His-MGF505–6R, pGL3-IFN-β-Luc, and pRL-TK, along with the indicated signaling molecules for 24 h, or treated with 1 μg/mL of poly(dA:dT) for 12 h. Subsequently, the cells were lysed for firefly and Renilla assays using the dual luciferase reporter gene assay kit, following the manufacturer’s protocol (Beyotime Biotechnology, Shanghai, China).

For the detection of mRNA levels of IFN-β and antiviral genes ISG54, ISG56, CXCL10, and TNF-α, HEK-293T and SK6 cells were transfected with either an empty vector or His-MGF505–6R, along with the indicated signaling molecules for 24 h, or treated with 1 μg/mL poly(dA:dT) for 12 h. For the detection of MGF505–6R transcriptional kinetics, porcine alveolar macrophages (PAMs) infected with 1 MOI of ASFV strain SXH1 were collected at 0 h, 6 h, 12 h, 24 h, and 36 h post-infection. Subsequently, total RNA was extracted using TRIpure reagent (Aidlab Technologies, Beijing, China), and 1 μg of RNA was reverse transcribed into cDNA using Hifair^®V one-step RT-gDNA digestion SuperMix (YEASEN, Shanghai, China). The cDNA was then subjected to qRT-PCR detection with Hieff^® qPCR SYBR Green Master Mix (Low Rox Plus) (YEASEN, Shanghai, China) according to the manufacturer’s protocol. The relative mRNA levels were normalized to GAPDH mRNA, referring to the 2-ΔΔCT method. All primers used for qPCR detection are listed in Supplementary Table 1 in the Supplementary Material .

For co-immunoprecipitation (co-IP) assays, HEK-293T or SK6 cells were pre-seeded into 6-well plates and cultured until they reached 70–80% confluence. Subsequently, the cells were transfected with the indicated plasmids for 24 h. Afterward, the cells were lysed with 150 μL of NP-40 lysis buffer (50 mM HEPES, pH 7.4, 200 mM NaCl, 1 mM EDTA, 1% NP-40) supplemented with a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China) for 40 min on ice. Then, 40 μL of supernatant from lysed cells was directly used for western blot assays with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after being mixed with 5× loading buffer and boiled at 95°C for 10 min. To test for co-immunoprecipitation (co-IP), the remaining 100 μL of supernatant was incubated with the indicated antibodies for 2 h. Then, 20 μL of protein A+G agarose gel (Beyotime Biotechnology, Shanghai, China) was added and incubated for an additional 1 h. Subsequently, the mixtures were thoroughly washed five times to remove nonspecific precipitates. The protein samples were separated using 12% SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the membrane was blocked with 5% skimmed milk in PBST buffer (PBS containing 0.1% Tween 20) for 1 h at 37°C or overnight at 4°C, and then incubated with the indicated primary antibodies for 1.5 h. After three washes, the membranes were incubated with horseradish peroxidase-coupled goat anti-mouse IgG or goat anti-rabbit IgG (1:5000, MBL, Japan) for an additional 40 min, followed by three washes. Finally, the protein bands were visualized using the ECL chemiluminescence system (Thermo Fisher Scientific, Waltham, USA).

For immunofluorescence the coverslips were placed in 24-well plates and treated with poly-L-lysine before seeding HEK-293T cells. Then the indicated plasmids were transfected into a 70–80% confluent monolayer for 24 h. Subsequently, the cells were fixed with 4% formaldehyde for 15 min at 37°C and permeabilized with 0.2% Triton X-100 for 20 min at 4°C. After being washed three times with PBST, the cells were blocked with 1% bovine serum albumin (BSA) for 1 h at 37°C. Following this, the indicated primary antibodies were added and incubated for 1.5 h at 37°C. Subsequently, the samples were washed before adding Alexa Fluor 555 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit antibodies, which were then incubated for 40 min. The nucleus were stained with DAPI for 10 min. After washing, the images were observed and captured using a confocal laser scanning microscope (Ti-U-Nikon, Tokyo, Japan).

Short hairpin RNA (shRNA) targeting STING was cloned into the pLKO.1 vector (pLKO.1-shSTING). To generate STING-knockdown cells, HEK-293T cells were transfected with pLKO.1-shSTING, along with the packaging plasmids psPAX2 and pMD2.G. After 48 h transfection, the recombinant lentiviruses were collected from the supernatant of the cultured cells. Subsequently, HeLa cells were transduced with the collected recombinant lentiviruses in the presence of polybrene, and the knockdown efficiency was assessed using western blot analysis.

The small interfering RNA (siRNA) targeting autophagy-related gene 7 (siRNA-ATG7) was designed and synthesized by Sangon (Shanghai, China). To generate ATG7-knockdown cells, HEK-293T cells were transfected with siRNA-ATG7 for 12 h and subsequently analyzed using western blotting. The sequences of siRNA-ATG7 are provided in Supplementary Table 2 .

The data were analyzed using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA) and are presented as the mean ± standard deviation (SD) from at least two independent experiments. Statistical analyses were conducted using an unpaired, two-tailed Student’s t-test, assuming unequal variance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant).

To identify the ASFV-encoded proteins that affect the type I interferon signaling pathway, we screened 29 viral proteins belonging to the MGF family using a dual luciferase reporter system in HEK-293T cells. The results indicated that several proteins from the MGF505 family significantly inhibited the activation of the IFN-β promoter induced by Sendai virus (SeV) ( Supplementary Figure 1 ). Among these proteins, MGF505–6R displayed the most pronounced antagonistic effects.

To further dissect the antagonistic mechanisms employed by MGF505–6R, which have not been reported previously, we conducted dual luciferase assays by transfecting pGL3-IFN-β-Luc, pRL-TK, along with His-MGF505–6R or empty vector (EV) plasmids into swine kidney-6 (SK6) cells, and using poly(dA:dT) as an IFN-β inducer. The results indicated that MGF505–6R measurably attenuated poly(dA:dT)-induced IFN-β promoter activation in a dose-dependent manner in SK6 cells ( Figure 1A ). Furthermore, the mRNA levels of IFN-β and antiviral genes were investigated in SK6 cells after transfection with the indicated plasmids and treatment with poly(dA:dT). The results showed that overexpression of MGF505–6R effectively reduced the mRNA levels of IFN-β and the antiviral genes ISG54, ISG56, CXCL10, and TNFα in SK6 cells ( Figure 1B ). To investigate whether MGF505–6R antagonizes type I interferon by targeting the cGAS-STING axis, a general cytosolic dsDNA sensing pathway, SK6 cells were transfected with pGL3-IFN-β-Luc, pRL-TK, Flag-cGAS, Flag-STING, and increasing doses of His-MGF505–6R plasmids, and then subjected to dual luciferase assays. The results showed that MGF505–6R inhibited cGAS-STING-mediated IFN-β promoter activation in SK6 cells in a dose-dependent manner ( Figure 1C ). Furthermore, we also discovered that MGF505–6R reduced cGAS-STING-mediated mRNA levels of IFN-β and the antiviral genes ISG54, ISG56, CXCL10, and TNF-α compared to the empty vector in SK6 cells ( Figure 1D ). To directly visualize the inhibitory effects of MGF505–6R on type I interferon responses, SK6 cells were transfected with His-MGF505–6R or an empty vector and then infected with eGFP-VSV before visualization by fluorescence microscopy. The results indicated that overexpression of MGF505–6R significantly enhanced eGFP-VSV replication in SK6 cells in the presence of poly(dA:dT). The average fluorescence intensity was then analyzed using ImageJ software ( Figure 1E ). Consistently, MGF505–6R also exerted antagonistic effects on the cGAS-STING signaling pathway in HEK-293T cells ( Supplementary Figure 2 ). These results demonstrate that MGF505–6R inhibits type I interferon responses by targeting the cGAS-STING signaling pathway.

We examined signaling molecules of the cGAS-STING, Toll-like receptor, and RIG-I-like receptor signaling pathways that lead to IFN-β production. The results indicated that the ectopic expression of MGF505–6R inhibited the activation of the IFN-β promoter in a dose-dependent manner by suppressing cGAS and STING but not TRIF, MAVS, TBK1, and IRF3 ( Figures 2A–F ). These results demonstrate that STING could be a potential target of MGF505–6R for counteracting the cGAS-STING signaling pathway. To further support this hypothesis, STING-knockdown HeLa cells were generated using shRNA and then subjected to dual luciferase assays. The results showed that MGF505–6R lost its inhibitory effects on poly(dA:dT)-induced IFN-β promoter activation in STING-knockdown HeLa cells ( Figure 2G ). To visualize the impact of MGF505–6R on virus replication in the absence of STING, wild-type and STING-knockdown HeLa cells were infected with eGFP-VSV while overexpressing MGF505–6R and then observed using fluorescence microscopy. The results indicated that MGF505–6R promoted the replication of eGFP-VSV in wild-type HeLa cells but did not affect viral replication in the absence of STING ( Figure 2H ). The average fluorescence intensity was calculated using Image J ( Figure 2I ). Taken together, these results demonstrate that MGF505–6R reduces type I interferon production by targeting STING protein.

Given that MGF505–6R impairs IFN-β responses by targeting STING, we wanted to investigate whether MGF505–6R directly interacts with STING. Specifically, we screened proteins on the cGAS-STING signaling pathway that were inhibited by MGF505–6R in the IFN-β luciferase assays. The results showed that MGF505–6R only co-precipitated with STING but not with cGAS, TBK1, IKKϵ, or IRF3 using anti-Flag agarose beads ( Figure 3A ) and anti-His agarose beads ( Figure 3B ). Furthermore, we observed interactions between MGF505–6R and endogenous STING in co-IP studies following overexpression of MGF505–6R in SK6 cells ( Figure 3C ). Consistently, colocalization between MGF505–6R and STING was observed in the cytoplasm following the overexpression of MGF505–6R and STING proteins in HEK-293T cells ( Figure 3D ). Together, all the data demonstrate that MGF505–6R interacts with STING to negatively regulate host antiviral immune responses.

Considering that MGF505–6R mainly interacts with STING to exert inhibitory effects on type I interferon responses, we wanted to investigate whether MGF505–6R affects the protein levels of STING. HEK-293T cells transfected with Flag-cGAS, Flag-STING, Flag-TBK1, and Flag-IRF3, along with increasing amounts of His-MGF505–6R plasmids, were analyzed using western blot and qRT-PCR. The results show that the overexpression of MGF505–6R significantly reduced the protein level of STING in a dose-dependent manner, while it had no impact on the protein levels of cGAS, TBK1, and IRF3. Meanwhile, the mRNA levels of cGAS, STING, TBK1, and IRF3 remained unaffected subsequent to the overexpression of MGF505–6R ( Figures 4A–D ). We further performed a cycloheximide (CHX) chase experiment to determine how MGF505–6R affects the endogenous STING protein levels. The results showed that overexpression of MGF505–6R significantly decreased STING protein levels compared to HeLa cells transduced with an empty vector after treatment with cycloheximide (CHX), a protein synthesis inhibitor ( Figure 4E ). These demonstrate that MGF505–6R affects the stability of STING. Furthermore, we detected the transcriptional kinetics of MGF505–6R and the STING protein levels in porcine alveolar macrophages (PAMs) during ASFV infection. The results showed that MGF505–6R was transcribed at 6 h post-infection, even earlier than E183L-encoded p54 protein ( Figure 4F ). The amounts of STING obviously decreased at 12 h post-infection, which was reasonably due to MGF505–6R expression ( Figure 4G ). To investigate the protein degradation pathways utilized by MGF505–6R to reduce the protein levels of STING, HEK-293T cells were transfected with His-MGF505–6R and Flag-STING plasmids. Subsequently, the cells were treated with the proteasome inhibitor MG132, the general caspase inhibitor Z-VAD-FMK, and the autophagosome inhibitor CQ individually. The results indicated that the degradation of the STING protein, mediated by MGF505–6R, was effectively rescued by the autophagosome inhibitor CQ but not by the proteasome inhibitor MG132 or the caspase inhibitor Z-VAD-FMK ( Figures 4H–J ). Consistent with this, we observed a dose-dependent increase in the conversion of LC3-I to LC3-II protein with increasing MGF505–6R ( Figure 4K ). To validate the findings, HEK-293T cells with knockdown in the autophagy-related gene (ATG7) were generated using siRNA targeting ATG7 (siRNA-ATG7) ( Figure 4L ). MGF505–6R did not facilitate the transformation of LC3-I to LC3-II and the consequent degradation of STING when ATG7 was knocked down ( Figure 4M ). In summary, these findings demonstrate that MGF505–6R facilitates the degradation of STING through the autophagy-lysosome pathway.

To further investigate which domains of MGF505–6R are involved in the suppression of type I interferon, the gene MGF505–6R was divided into two segments, MGF505–6R-N (1–260AA) and MGF505–6R-C (261–526AA), and subcloned into the pTRIP-CMV vector with a Flag tag, as depicted in Figure 5A . Dual luciferase assays were conducted following the ectopic expression of His-MGF505–6R, Flag-MGF505–6R-N, and Flag-MGF505–6R-C plasmids separately in HEK-293T cells. The results indicated that the MGF505–6R-N domain, rather than MGF505–6R-C, was accountable for the inhibition of poly(dA:dT)-stimulated IFN-β promoter activation ( Figure 5B ). Consistently, MGF505–6R-N also exhibited antagonistic effects on cGAS-STING-mediated IFN-β reporter activation ( Figure 5C ). Furthermore, co-IP with anti-HA agarose beads demonstrates that MGF505–6R and MGF505–6R-N, but not MGF505–6R-C, could co-precipitate with STNG ( Figure 5D ). Consistently, the degradation of STING in HEK-293T cells was mediated by MGF505–6R-N rather than MGF505–6R-C ( Figure 5E ). Collectively, the essential domain involved in inhibiting type I IFN responses was MGF505–6R-N. To further explore the domain of STING responsible for its interaction with MGF505–6R, the domains of STING were truncated and constructed as depicted in Figure 5F . Co-IP experiments were performed in HEK-293T cells following the ectopic expression of His-MGF505–6R, along with truncation plasmids of Flag-STING-TM, Flag-STING-NT, Flag-STINGΔCTT, Flag-STINGΔTM, and Flag-STING. The results indicated that STING and STINGΔTM, but not STING-TM, STING-NT, or STINGΔCTT truncations, were able to co-precipitate with MGF505–6R when using anti-Flag agarose beads ( Figure 5G ). The data demonstrate that the STING CTT domain plays a crucial role in the interaction with MGF505–6R.

Ubiquitination plays a crucial role in regulating the STING-mediated signaling pathway. To assess the impact of MGF505–6R on STING polyubiquitination, ubiquitin and ubiquitin mutants either retained a single lysine residue (KO) or retained all lysine residues except one (KR), and mutating all lysine residues to arginine (AKR) were constructed ( Figure 6A ). HEK-293T cells transfected with His-MGF505–6R exhibited a significant reduction in STING polyubiquitination, both in the presence and absence of MG132, compared to cells transfected with an empty vector. However, MGF505–6R did not impact the polyubiquitination of STING when all lysine residues were mutated to arginine (AKR) ( Figure 6B ). To examine the polyubiquitin chains linked to STING that were affected by MGF505–6R, co-IP assays were performed on HEK-293T cells transfected with ubiquitin and mutants containing a single lysine residue at positions 6, 11, 27, 29, 33, 48, and 63. The results indicated that MGF505–6R clearly facilitated the elimination of K63-linked ubiquitination from STING while not affecting ubiquitination associated with any other linkage ( Figure 6C ). Moreover, MGF505–6R failed to reduce STING polyubiquitination when the lysine residue at position 63 was substituted with arginine (K63R) ( Figure 6D ). The necessity of K63-linked polyubiquitination for the oligomerization of STING and its recruitment to TBK1 has been documented (30). Our further investigation revealed that MGF505–6R overexpression significantly inhibited the interaction between HA-STING and Flag-STING in HEK-293T cells compared to the empty vector ( Figure 6E ). Consistently, the ectopic expression of MGF505–6R reduced the interaction between STING and TBK1 ( Figure 6F ). Additionally, MGF505–6R overexpression dose-dependently attenuated poly(dA:dT)-induced aggregation of endogenous STING in HeLa cells ( Figure 6G ). In general, MGF505–6R inhibits the K63-linked polyubiquitination of STING, thereby hindering the oligomerization of STING and the recruitment of STING to TBK1.

Upon activation of the cGAS-STING-TBK1 axis, phosphorylation and nuclear translocation of IRF3 are initiated, leading to the generation of type I interferon and subsequent expression of antiviral genes. Our study revealed that overexpression of MGF505–6R notably suppressed the poly(dA:dT)-induced phosphorylation of IRF3 in HEK-293T cells ( Figure 7A ). Consistently, MGF505–6R impaired IRF3 phosphorylation in a dose-dependent manner, which was induced by overexpression of cGAS-STING or STING alone in HEK-293T cells ( Figures 7B, C ). To support the findings, the subcellular fractions of the nucleus and cytoplasm were isolated and underwent immunoblot analysis after transfection with increasing doses of His-MGF505–6R. The results indicated that MGF505–6R reduced the levels of IRF3 in the nucleus in a dose-dependent manner following poly(dA:dT) treatment in HEK-293T cells ( Figure 7D ). Furthermore, we observed an inhibition of poly(dA:dT)-induced nuclear translocation of IRF3 in HEK-293T cells by MGF505–6R overexpression using confocal microscopy ( Figure 7E ). The nuclear IRF3 was quantified using ImageJ ( Figure 7F ). The data collectively demonstrate that MGF505–6R suppresses the phosphorylation and nuclear translocation of IRF3, leading to a subsequent impairment in the production of IFN-β and antiviral genes.