Pig STING protein K61R mutant and application thereof

Abstract

The invention discloses a pig STING protein K61R mutant and application thereof, the mutant is obtained by mutating the 61th lysine of the pig STING protein into arginine, and the amino acid sequence of the pig STING protein is as shown in SEQ ID NO. 1. According to the invention, the species-specific key site K61 of the pig STING protein is identified for the first time, the site is unique to pigs and is different from other species, and by regulating the protein stability and downstream signal transduction, a key negative regulation effect on virus replication of ASFV, PRV, HSV-1 and the like is achieved; meanwhile, the K61 site can be directly used as a core molecular marker and a targeting site for pig antiviral breeding, and important support is provided for improving the research efficiency of disease-resistant breeding.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical technology, and particularly relates to a pig STING protein K61R mutant and application thereof in broad-spectrum anti-viral infection. BACKGROUND

[0002] It is known that natural immunity is the first line of defense against pathogenic microorganism infection, and through recognizing pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP) common to pathogens and self, activates natural immune response, produces various cytokines including interferon (IFN) and inflammatory factors, and plays an anti-infection role. The pattern recognition receptor (PRR) of natural immunity includes the Toll-like receptor (TLR), RIG-I-like receptor (RLR), NOD-like receptor (NLR), C-type lectin-like receptor (CLR) and cytoplasmic DNA receptor (CDR) families. As an important cytoplasmic DNA receptor signaling pathway, cGAS-STING is involved in natural immune response caused by viruses, bacteria infection and damage, and is one of the foundations for activating subsequent adaptive immunity.

[0003] STING protein (stimulator of interferon genes) as a linker protein in the cGAS-STING signaling pathway, its core function is to activate the production of interferon (IFN) and inflammatory cytokines. Not only that, STING protein has a variety of non-interferon (IFN) cell functions, including autophagy, cell death (such as apoptosis, necrosis, pyroptosis and lysosome-dependent cell death), ER stress, metabolic reprogramming, etc. cGAS-STING-IFN plays a direct role in recognizing DNA viruses and retroviruses (HIV) infection and antiviral infection. Common DNA viruses include herpes simplex virus (HSV-1), poxvirus (VV), cytomegalovirus (CMV), porcine circovirus (PCV2), porcine pseudorabies virus (PRV) and African swine fever virus (ASFV), as well as chicken Marek's disease virus (MDV) and avian adenovirus (FAdV-4). In addition, cGAS also shows an indirect anti-RNA virus effect, mainly due to the cell mitochondrial and nuclear DNA damage caused by RNA virus replication in cells, leading to mitochondrial DNA (mtDNA) and nuclear DNA leakage into the cytoplasm, thereby activating the cGAS-STING-IFN signaling pathway, so cGAS-STING shows a broad-spectrum antiviral effect. Correspondingly, when studying the cGAS-STING-IFN signaling pathway in pigs and chickens, it was found that STING signaling had a broad-spectrum antiviral effect. There are also reports that STING downstream NF-κB signaling is involved in antiviral effects. As mentioned earlier, STING activation triggers cell functions independent of IFN signaling, including autophagy. STING-activated autophagy also has an antiviral effect, and this antiviral effect does not depend on IFN.

[0004] Post-translational modification of proteins includes phosphorylation, glycosylation, methylation, acetylation, palmitoylation and ubiquitination, etc. These modifications can change the structure, stability, activity, localization of proteins and affect the interaction with other proteins or small molecule compounds. Ubiquitination modification is one of the types of post-translational modification of proteins that was discovered and studied earliest. Ubiquitin molecules are covalently bound to specific lysine (K) residues of target proteins under the catalysis of various enzymes. Target proteins labeled by ubiquitin molecules will have different fates due to different types of ubiquitination or ways of ubiquitin chain connection. Ubiquitination modification of proteins is involved in various forms of life activities, including protein activity and function, protein degradation and cell cycle and differentiation.

[0005] The activation and inactivation processes of cGAS-STING involve multiple post-translational modifications. For STING protein, palmitoylation at cysteine 91 (Cys91) site is critical for its functional regulation. Palmitoylation modification changes the hydrophobicity and membrane binding property of STING protein. Unpalmitoylated STING is mainly localized in endoplasmic reticulum, while after palmitoylation modification, STING can be transferred from endoplasmic reticulum to Golgi and produce a non-cGAS-dependent activation effect. On the Golgi, TBK1 promotes the recruitment of IRF3 and activates the subsequent type I interferon (IFN) signal by adding a phosphate group to S366 of the STING protein. After activation, STING is degraded through autophagy, recycling endosome or ubiquitin proteasome pathway to avoid persistent activation of immune response. Ubiquitination modification is involved in the negative feedback regulation of STING in multiple forms. In humans and mice, various E3 ligases such as RNF5, TRIM29, TRIM13 mediate K48-linked ubiquitination of STING, degrading STING through proteasome. In addition, K63-linked polyubiquitination of STING mediates its binding to autophagy receptors and transport to autolysosomes and recycling endosomes, also playing a role in negative feedback regulation. On the other hand, K63-linked polyubiquitination is also crucial for STING protein-mediated type I interferon (IFN) activity. Various E3 ligases including RNF115, TRIM56 and TRIM10 promote STING translocation to Golgi, dimerization and subsequent anti-viral infection signal by enhancing STING K63-linked ubiquitination. In short, ubiquitination modification of STING plays a complex regulatory role in its activation of multiple signals.

[0006] Both STING and cGAS are highly conserved host molecules, which can be traced back to unicellular protists Brevi corythell a Monosiga brevicollis The sea anemone Nematostella vectensis Nematostella vectensisSTING not only responds to 3'3'-cGAMP, but also responds to 2'3'-cGAMP in vertebrate cells. Vertebrates have evolved IFN, whose STING downstream signals produce IFN responses. Although very conservative in evolution, STING shows species specificity in different species. For example, unlike invertebrates, vertebrates have evolved IFN; in response to this, their STING has evolved a CTT domain to produce IFN. Human and mouse STING also show species specificity; several non-nucleotide small molecule activators, CMA, FAA and DMXAA, can well activate mouse STING, but cannot activate human STING, and the specific mechanism is not clear. Our team has also found species differences in pig and chicken STING; the subtle differences in the pLxIS motif on the CTT of the two STINGs lead to differences in recognizing downstream transcription factors IRF.

[0007] For post-translational modification of STING protein, reports in different species are also different, especially ubiquitination modification. Unlike E1 and E2, which are highly conserved among different species, the homology of E3 ligase decreases with increasing evolutionary distance. The E3 ligase that mediates the ubiquitination modification of STING has a certain species specificity. Mouse TRIM30a promotes the K48-linked polyubiquitination of STING at K150, but there is no complete homologous ortholog protein of this protein in humans and pigs. In addition, in fish and humans, K48-linked polyubiquitination is often reported to occur at K150 or homologous sites, while the sites of mouse STING-mediated ubiquitin-proteasome degradation are reported to occur at the C-terminal of the protein, such as K370 and K338 / K347, suggesting that the site preference of the degradation mechanism is different between different species.

[0008] STING, as a key antiviral protein in the DNA receptor pathway of innate immunity, has been widely studied in the biomedical field. Currently, the antiviral effect of cGAS-STING signaling in livestock and poultry animals is also widely recognized. However, the species specificity of the cGAS-STING signaling pathway is still lacking. In pigs, the conservation of the function of the antiviral protein in pig subspecies is a potential direction for the selection of antiviral pigs. SUMMARY

[0009] The present application aims to solve the technical problems, overcome the shortcomings of the prior art and provide a pig STING protein K61R mutant and its application in broad-spectrum antiviral infection.

[0010] One of the purposes of the present application is to provide a pig STING protein mutant, characterized in that the mutant is a lysine at position 61 of the pig STING protein mutated into arginine, and the amino acid sequence of the pig STING protein is shown in SEQ ID NO. 1.

[0011] The present application finds that K61 of STING is a key negative regulator by positive and negative tests through single mutation or single reservation of all lysines K on pig STING, and after mutation of K61, the expression of STING protein and the anti-virus signal are significantly enhanced. Sequence comparison combined with cloning and mutation of multiple species of STING finds that STING K61 has pig species specificity in negative regulation of its own protein and signal. At the same time, RNF5 and USP20 are identified as E3 ligase and deubiquitinase mediating K48-linked polyubiquitination on STING K61, which respectively play negative and positive regulation roles after activation of STING. Since the cGAS-STING signal is a key signal pathway of natural immunity against viruses, the discovery of the pig STING specific K61 site and its degradation ubiquitination E3 ligase and deubiquitinase provides an important target for pig disease resistance breeding and anti-virus small molecule drug design.

[0012] The second purpose of the present application is to provide a polynucleotide molecule encoding the pig STING protein K61R mutant as described above.

[0013] The third purpose of the present application is to provide a vector comprising the polynucleotide molecule as described above.

[0014] The fourth purpose of the present application is to provide a host cell comprising the vector or the chromosome of the host cell integrated with the polynucleotide molecule as described above.

[0015] The fifth purpose of the present application is the application of the pig STING protein K61R mutant, the polynucleotide molecule, the vector or the host cell as described above in broad-spectrum anti-virus infection.

[0016] In the above-mentioned application, the pig STING protein K61R mutant, the polynucleotide molecule, the vector or the host cell are used in the preparation of a medicine for preventing and / or treating pig disease virus infection.

[0017] In the above-mentioned application, the pig disease virus includes but is not limited to African swine fever virus (ASFV), porcine pseudorabies virus (PRV), vesicular stomatitis virus (VSV), herpes simplex virus-1 (HSV-1).

[0018] In the above application, by site-directed mutagenesis of the 61st amino acid of the STING protein, changing it from lysine (K) to arginine (R), it is backfilled into monkey epithelial cells and pig alveolar macrophage cell lines with knockout STING genes, to inhibit the replication of African swine fever virus and pseudorabies virus and other pig disease viruses.

[0019] The fifth object of the present application is the application of the pig STING protein K61R mutant, polynucleotide molecule, vector or host cell as described above in breeding virus-resistant breeding pigs.

[0020] The present application provides a key site K61 of pig STING protein degradation and antiviral species specificity, which is different from other species, and plays a key negative regulation role in protein stability and subsequent signal transduction and anti-ASFV, PRV and HSV-1 and other virus replication, and STING K61 site can be used as a molecular marker and target for antiviral breeding, which is of great significance to improve the research level of antiviral breeding, and small molecule regulators (such as RNF5 inhibitors) targeting this site have become the development direction of new antiviral drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 3 is a diagram of screening results for the degradation of STING after activation in HEK293T cells and 3D4 / 21 cells.

[0022] Figure 1 In the above application, by site-directed mutagenesis of the 61st amino acid of the STING protein, changing it from lysine (K) to arginine (R), it is backfilled into monkey epithelial cells and pig alveolar macrophage cell lines with knockout STING genes, to inhibit the replication of African swine fever virus and pseudorabies virus and other pig disease viruses.

[0023] Figure 2 Figure 3 is a diagram of screening results for the degradation of STING after activation in HEK293T cells and 3D4 / 21 cells. - / - Figure 3 is a diagram of screening results for the degradation of STING after activation in HEK293T cells and 3D4 / 21 cells.

[0024] Figure 2A, B, C and D are the results of luciferase reporter assay to detect the difference of ISRE and NF-κB promoter activity activated by pSTING and each K-R mutant transfection HEK293T cells; E is the Westen blotting to detect the difference of pSTING and K61R mutant expression in STING - / - 3D4 / 21 cells complemented with pSTING and K61R mutant, the difference of downstream related protein expression after 2’3’-cGAMP stimulation; D is the qRT-PCR to detect the difference of downstream related gene expression after 2’3’-cGAMP stimulation in STING - / - 3D4 / 21 cells complemented with pSTING and K61R mutant, the difference of downstream related protein expression after 2’3’-cGAMP stimulation; D is the qRT-PCR to detect the difference of downstream related gene expression after 2’3’-cGAMP stimulation in STING

[0025] Figure 3 The results of the role of STING K61 in anti-ASFV and PRV infection.

[0026] Figure 3 A, B, C and D are the results of luciferase reporter assay to detect the difference of ISRE and NF-κB promoter activity activated by pSTING and each K-R mutant transfection HEK293T cells; E is the Westen blotting to detect the difference of pSTING and K61R mutant expression in STING - / - MA104 cells complemented with pSTING and corresponding mutant, then infected with ASFV-GFP, flow cytometry to detect the positive cell rate of ASFV infection in different groups; B is the statistical chart of flow cytometry results; C is the Westen blotting to detect the expression of p72 and p30 protein of ASFV after the same treatment as A; D and E are the qRT-PCR to detect the expression of PRV gD gene in cells (D) and plaque assay to detect the virus titer of PRV in cell supernatant (E) after MA104 cells complemented with pSTING and corresponding mutant were infected with PRV. Figure 3 A, B, C and D are the results of luciferase reporter assay to detect the difference of ISRE and NF-κB promoter activity activated by pSTING and each K-R mutant transfection HEK293T cells; E is the Westen blotting to detect the difference of pSTING and K61R mutant expression in STING - / - 3D4 / 21 cells complemented with pSTING and K61R mutant, the difference of downstream related protein expression after 2’3’-cGAMP stimulation; D is the qRT-PCR to detect the difference of downstream related gene expression after 2’3’-cGAMP stimulation in STING

[0027] Figure 4 The results of the role of STING K61 in anti-ASFV and PRV infection.

[0028] Figure 4A, B, C and D are the results of Westen blotting detection of ubiquitination of pSTING at K61 site, potential sites of polyubiquitination of pSTING at K48 site, uniqueness of K48 ubiquitination of pSTING at K61 site, and expression of purified UBA1, UBE2D1, Ub-K48O, GST, GST-STING and GST-STING K61R proteins in E. coli, respectively; E and F are the results of Westen blotting detection of K48 ubiquitination of pSTING in vitro and K48 ubiquitination of purified pSTING and K61R mutant proteins, respectively.

[0029] Figure 5 Figure 4 is a diagram of screening of ubiquitination of different species of STING and K61 homologous site mutants.

[0030] Figure 5 A is a diagram of sequence comparison of STING K61 site of nine species of human, green monkey, mouse, rat, pig, cow, sheep, chicken and zebrafish; B-D are the results of Westen blotting detection of expression of self and downstream related proteins, Luciferase assay detection of activity of ISRE promoter, and qRT-PCR detection of transcription of downstream IFN-β and CXCL10 genes after transfection of HEK293T cells with STING and corresponding K61 homologous site mutants of green monkey, mouse, pig, cow and chicken, respectively; E-I are the results of Westen blotting detection of changes in total ubiquitination and K48 site connected ubiquitination of STING after mutation of K61 homologous site of different species of STING after transfection of HEK293T cells with STING and corresponding K61 homologous site mutants of pig (E), green monkey (F), mouse (G), cow (H) and chicken (I), respectively; J is a diagram of construction of two substitution mutants of bovine STING; K and L are the results of Westen blotting detection of effects of total ubiquitination (K) and K48 site connected ubiquitination (L) of bovine STING after mutation of two sites.

[0031] Figure 6 Figure 5 is a diagram of screening results of E3 ligase mediating K48 site connected polyubiquitination of pSTING at K61.

[0032] Figure 6A, B and C are the results of Westen blotting detecting the influence of four E3 ubiquitin ligases on pSTING expression (A), total ubiquitination of pSTING (B) and K48-linked ubiquitination (C); D is the result of laser confocal detecting the subcellular co-localization of pSTING and four E3 ligases; E is the result of Co-immunoprecipitation detecting the interaction between endogenous STING and RNF5 at different times after HSV-1 infection.

[0033] Figure 7 Figure 4 is a result chart of the influence of RNF5 ubiquitination modification of pSTING and downstream signal.

[0034] Figure 7 A-C are the results of luciferase reporter gene detecting the activity of pSTING downstream ISRE and NF-κB promoters (A), Westen blotting detecting the expression of pSTING and downstream related proteins (B), and qRT-PCR detecting the transcription of pSTING activated antiviral genes (C) in HEK293T cells transfected with pSTING and different concentrations of RNF5; D is the result of Westen blotting detecting the influence of RNF5 on the expression of pSTING and K61 mutant proteins; E is the result of Coomassie blue staining after SDS-PAGE electrophoresis detecting the expression of purified RNF5, Ub-K48O and Ub4-K48O proteins in E. coli; F is the result of GST-pulldown assay detecting the interaction between in vitro purified pSTING protein and RNF5 protein; G is the result of Westen blotting detecting the influence of in vitro RNF5 on K48-linked ubiquitination of pSTING; H is the result of Westen blotting detecting the influence of RNF5 on each type of ubiquitination of pSTING; I is the result of Westen blotting detecting the influence of RNF5 on K29, K33 and K48-linked ubiquitination of pSTING K61R mutant; J is the result of Westen blotting detecting the influence of RNF5 on ubiquitination of pSTING K61O mutant.

[0035] Figure 8 Figure 6 is a diagram of RNF5 and STING interaction domain analysis.

[0036] Figure 8Figure 1. The schematic diagram of the construction of pSTING and its eight truncations (A), the interaction of RNF5 and pSTING and its eight truncations (B and C), the key domains of pSTING interacting with RNF5 (D), the schematic diagram of the construction of RNF5 and its four truncations (E), the interaction of pSTING and RNF5 and its four truncations (F and G), and the key domains of RNF5 interacting with pSTING (H).

[0037] Figure 9 Figure 2. The effect of RNF5 knockout on the expression of pSTING protein and downstream signals.

[0038] Figure 9 Figure 3. The effect of RNF5 knockout on the transcription of anti-virus genes activated by STING (A), the effect of RNF5 knockout on the degradation of STING and the expression of downstream related proteins after the activation of STING (B), the effect of 3-MA and NH4Cl on the degradation of STING in the presence or absence of RNF5 (C and D), the effect of RNF5 knockout on the ubiquitination modification of STING after the activation of STING (E), the effect of RNF5 knockout on the anti-virus gene transcription activated by STING (F), and the effect of RNF5 knockout on the expression of pSTING and downstream related proteins (G). - / - Figure 4. The effect of RNF5 and TM2 knockdown on the activity of ISRE and NF-κB promoters after the activation of pSTING (F), the effect of RNF5 and TM2 knockdown on the transcription of anti-virus genes activated by pSTING (G), and the effect of RNF5 and TM2 knockdown on the expression of pSTING and downstream related proteins (H).

[0039] Figure 10 Figure 5. The screening of deubiquitinating enzymes for removing the polyubiquitination of pSTING K48 linkage.

[0040] Figure 10In the figure, A shows the effect of nine deubiquitinizing enzymes on the K48-linked ubiquitination of pSTING detected by Western blotting; B shows the effect of nine deubiquitinizing enzymes on the K48-linked ubiquitination of pSTING detected by Western blotting under RNF5 overexpression.

[0041] Figure 11 The figure shows the effect of USP20 knockout of pSTING protein expression and downstream signaling.

[0042] Figure 11 In the figures, A and B represent the effects of USP20 knockout on the self-degradation and downstream protein expression of STING after activation, as detected by Western blotting (A), and the effect of USP20 knockout on the transcription of antiviral genes activated by STING, as detected by qRT-PCR (B). C represents the effect of USP20 knockout on ubiquitination modification after STING activation, as detected by Western blotting. 1D-H represent the effects of USP20 knockout on STING activation-mediated anti-HSV-1 (D) and VSV (F) infection, as observed by fluorescence, and the viral titers of HSV-1 (E), VSV (G), and PRV (H) in the supernatant of infected cells in different treatment groups were determined by plaque assay.

[0043] Figure 12 Genotyping results for STING, RNF5, and USP20 knockout cells.

[0044] Figure 12 In the above, A is... Figure 3 The STING used in - / - Genotype comparison diagram of MA104 cells and WT cells; B and C are the above. Figure 9 The two RNF5 plants used in - / - Genotypic comparison of 3D4 / 21 cells and WT cells; D and E are... Figure 11 The two USP20 plants used in - / - Genotypic comparison of 3D4 / 21 cells and WT cells. Detailed Implementation

[0045] The present application provides a STING ubiquitination modification site mutant, which is mutated at the 61st amino acid residue, i.e. the 61st lysine (Lys, K) is mutated to arginine (Arg, R). The present application research finds that during viral infection, pig STING is activated to recruit E3 ubiquitin ligase RNF5, which specifically assembles K48-linked polyubiquitin chains at the K61 site, and then is recognized by the 26S proteasome and degraded, which negatively regulates the protein stability of STING, and significantly weakens its antiviral signal function. Moreover, although the sequence of this site is relatively conserved in mammals, it only presents this phenotype in pigs. For the modified site on STING, through gene editing technology, multiple gene synergistic editing of pig STING related genes can cultivate pigs resistant to multiple viral infections. Therefore, the discovery of the specific site of pig STING mediated protein degradation provides an important key target for antiviral pig breeding, and provides a new idea for preventing and controlling pig viral infection.

[0046] Material Description: (1) Plasmid, cell line and strain Plasmids: The eukaryotic expression plasmids pcDNA-DEAST47-pcGAS-2HA, pEGFP-C1-pSTING, pEGFP-C1, pmCherry-C1, pmCherry-C1-pSTING, p3xFlag-CMV-7.1, p3xFlag-CMV-7.1-pSTING, pCAGGS-2HA, pCAGGS-Myc, pX458-GFP, pET32a-12His, pCold I-GST and mammalian IFN-β firefly luciferase reporter plasmid (IFN-β-Fluc), ISRE firefly luciferase reporter plasmid (ISRE-Fluc), β-actin Renilla luciferase reporter plasmid (β-actin-Rluc) were all preserved by the Animal Infection and Immunity Laboratory of Yangzhou University. Among them, pEGFP-C1 (P0134), pmCherry-C1 (P0151), p3xFlag-CMV-7.1 (P0438), pCAGGS (P0165), pET32a (P0033), pCold-GST (P0337) empty vector plasmids were purchased from Miro Biosciences. For the three different vectors of STING, the nucleotide sequence of pig STING (GenBank: NM_001142838.1) was introduced into Benchling, and different vector amplification primers were designed. The primer sequences are shown in Table S1. The pig cDNA was used as a template for PCR amplification using gold MIX (green) enzyme. After amplification, the gel was recovered. The eukaryotic expression vectors pEGFP-C1, pmCherry-C1 and p3xFlag-CMV-7.1 were double digested with restriction endonuclease BglII / EcoRI, BglII / EcoRI and EcoRI / SalI, respectively. The digested products were gel recovered, and the recovered target fragments were cloned with the corresponding double-digested linear vectors using 2x MultiF Seamless Assembly Mix. pX458-GFP, pET32a-12His and luciferase reporter plasmids were from Youbao Biosciences.

[0047] For other expression plasmids, total RNA was extracted from Vero cells, Raw264.7 cells, MDBK cells and 3D4 / 21 cells using TRIpure reagent, and the open reading frame (ORF) of monkey STING (XM_008014636), mouse STING (XM_017317994), bovine STING (NM_001046357), pig RNF5 (NM_001123224), pig TRIM29 (XM_021062976), pig TRIM13 (XM_021065576), pig TRIM7 (XM_021083408), pig USP20 (XM_021070139), pig USP35 (XM_013979184), pig USP44 (XM_021078961), pig USP49 (XM_021098683), pig CYLD (XM_021094185), pig OTUD5 (XM_021080668), pig EIF3F (XM_021062272), pig RNF26 (XM_005667432), pig UBA1 (XM_021080354), pig UBE2D1 (NM_001078674), etc. was obtained by RT-PCR amplification, and the primer sequences used are shown in Table S1. The PCR products were respectively cloned into the EcoR I and Sal I enzyme sites of the p3xFlag-CMV-7.1 vector, the EcoR I and EcoR V enzyme sites of the pCAGGS-Myc / 2HA vector, the Bgl II and EcoRI enzyme sites of the pmCherry-C1 vector, the EcoR I and Xho I enzyme sites of the pET32a-6His vector, and the XhoI and EcoR I enzyme sites of the pCold I-GST vector. The Ub-K48 fragment was amplified by PCR from the commercial plasmid pRK5-6xHis-Ubiquitin-K48 (P37539) and ligated into the pET32a vector to construct a prokaryotic expression plasmid. All PCR products were ligated into the vector plasmid by homologous recombination with 2xMultiF Seamless Assembly Mix. For mutant expression plasmids, mutant PCR primers for multiple species of STING were designed based on the QuickChange primer design method (https: / / www.agilent.com) (see Table S1), and p3xFlag-CMV-pig / monkey / mouse / cattle STING or pmCherry-C1-pig STING was used as a template for mutant PCR using KODplus neo polymerase. After Dpn I digestion, the PCR product was transformed into competent DMT Escherichia coli, and the resulting mutants were sequenced and verified.

[0048] Cell lines: Porcine alveolar macrophage cell line (3D4 / 21), porcine primary alveolar macrophage (PAM), human embryonic kidney 293T cells (HEK293T), rhesus monkey kidney cells (MA104) were preserved by the Animal Infection and Immunity Laboratory of Yangzhou University. Among them, D4 / 21 cells (ATCC cat# CRL-2843), HEK293T cells (ATCC cat# CRL-3216), MA104 cells (ATCC cat# CRL-2378.1), and PAM are primary alveolar macrophages isolated from experimental pigs.

[0049] Strains: E. coli DH5a competent bacteria were stored by the Animal Infection and Immunity Laboratory of Yangzhou University.

[0050] (2) Reagents and viruses Cell culture medium DMEM, RPMI were purchased from Hyclone, USA. Fetal bovine serum was purchased from Beijing Zhongsheng Aobang Biotechnology Co., Ltd. Penicillin-streptomycin double antibody (100x, P1400) was purchased from Beijing Solayebio Technology Co., Ltd. TRIpure Reagent was purchased from Beijing Aidley Biotechnology Co., Ltd. Lipo2000 transfection reagent was purchased from Shanghai Thermo Fisher Company. High-fidelity enzyme TSINGKE TSE101 Gold Mix (green) (TSE102), pClone007 Versatile Simple Vector Kit (TSV-007VS) were purchased from Beijing Qikeng Biotechnology Co., Ltd. Dual Luciferase Reporter Assay Kit (DL101-01), Reverse Transcription Kit (HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper), R312-01), Gel Recovery / DNA Purification Kit (DC301-01), Plasmid Extraction Kit (DC201-01), Universal High Sensitivity Dye Method Quantitative PCR Detection Kit (Q711) and 180 kDa Prestained Protein Marker (MP102-01) were purchased from Nanjing Novozyme Biotech Co., Ltd. KOD-Plus-Neo high-fidelity DNA polymerase (KOD-401) was purchased from Oriental Yeast (Shanghai) Biotechnology Co., Ltd. Restriction endonuclease DpnI (R0176V), BglII (R0144V), XhoI (R0146V), EcoRI (R0101V), BbsI (R3539S), EcoRV (R3195V), SalI (R0138V) were purchased from NEB (Beijing) Co., Ltd. Homologous recombination enzyme 2XMultiF Seamless Assembly Mix (RK21020) was purchased from Wuhan Aiboteke Biotechnology Co., Ltd. PolyI:C-LMW, 2'3'-cGAMP and poly dA:dT were purchased from InvivoGen Company. Protein A / G PLUS-Agarose Beads were purchased from Santa Cruz Biotechnology Company. Annexin V-FITC / Propidium Iodide (PI) Apoptosis Detection Kit was purchased from Beckton-Dickinson Company, USA. T4 DNA ligase was purchased from Thermo Fisher Scientific Company, USA. 3-MA (HY-19312), MG132 (HY-13259) and NH4Cl (HY-Y1269) were purchased from MedChemExpress (MCE).4',6-diamidino-2-phenylindole (DAPI) staining solution (C1005), His-tag protein purification kit (P2226) and GST-tag protein purification kit (P2262) were purchased from Shanghai Biyun Tian Biotech Co., Ltd. Low-melting-point agarose and cycloheximide (CHX, C4859) were purchased from Sigma Aldrich, Germany. All other chemicals were of analytical grade and purchased from commercial channels.

[0051] Rabbit monoclonal antibodies: HA (3724S), LC3I / II (3868), cleaved caspase-3 (Asp175) (9664S), TBK1 (3504S), p-TBK1 (5483S, Ser172), IRF3 (11904S), FLAG (14793), GFP (2956) and β-actin (5057) were purchased from Cell Signaling Technology, USA. Phospho-IRF3 (Ser396) rabbit monoclonal antibody (MA5-14947) and DyLight™ 488 labeled cross-adsorbed goat anti-rabbit IgG (H+L) (35553) were purchased from Thermo Fisher Scientific, USA. Phospho-STAT1-Y701 rabbit monoclonal antibody (AP0054) was purchased from Wuhan Aibiotek Biological Technology Co., Ltd. mCherry rabbit polyclonal antibody (ab183628) and Alexa Fluor® 568 labeled goat anti-rabbit IgG H&L (ab175471) were purchased from Abeam, UK. Rabbit polyclonal antibodies: Myc (16286-1-AP), STING (19851-1-AP), ATG5-ATG12L (10181-2-AP), ubiquitin (10201-2-AP) and mouse monoclonal antibody GAPDH (60004-1-Ig) were purchased from Wuhan Sanying Biotech Co., Ltd. Rabbit polyclonal antibody RNF5 (abs118428) was purchased from Aibisheng Biological. Mouse monoclonal antibodies: HA (HT301-01), GFP (HT801-01), FLAG (HT201-01) and GST (HT601-01) were purchased from Quansikang Biotech Co., Ltd. HRP labeled high cross-adsorbed goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) secondary antibodies were purchased from Shengong Bioengineering (Shanghai) Co., Ltd. Mouse anti-p30 monoclonal antibody and anti-p72 monoclonal antibody were prepared and stored by the Animal Infection and Immune Laboratory of Yangzhou University.

[0052] Vesicular stomatitis virus (VSV-GFP), herpes simplex virus-1 (HSV-1-GFP), and pseudorabies virus (PRV, Bartha K61 strain, GenBank accession number JF797217) were all preserved in the Animal Infection and Immunology Laboratory of Yangzhou University. All ASFV infection experiments were performed in the Animal Biosafety Level 3 (ABSL-3) laboratory of Yangzhou University, which was approved by the Ministry of Agriculture and Rural Affairs. The ASFV YZ-1 strain (GenBank: ON456300.2) and the African swine fever virus ASFVΔMGF100-1R (ASFV-GFP) strain were used. Among them, the African swine fever virus ASFVΔMGF100-1R was based on the isolated ASFV YZ-1 strain (https: / / doi.org / 10.1186 / s44149-024-00130-1), with a gene deletion of MGF100-1R, and the obtained ASFV-GFP strain. The above strains were preserved in the Yangzhou University Animal Biosafety (ABSL-3) laboratory.

[0053] The remaining materials and reagents mentioned in the present application can be purchased from domestic and foreign commercial channels by the public, and will not be described one by one here.

[0054] Table S1. Amplification primer sequences and mutant gene PCR amplification primer sequences Primers Sequence (5'-3') <![CDATA[pEGFP-C1-pSTING-FpEGFP-C1-pSTING-RpmCherry-C1-pSTING-FpmCherry-C1-pSTING-RFLAG-pSTING-FFLAG-pSTING-RpSTING K61R-FpSTING K61R-RpSTING K112R-FpSTING K112R-RpSTING K150R-FpSTING K150R-RpSTING K187R-FpSTING K187R-RpSTING K236R-FpSTING K236R-RpSTING K289R-FpSTING K289R-RFlag-moSTING-FFlag-moSTING-RmoSTING K61R-FmoSTING K61R-RFlag-mSTING-FFlag-mSTING-RmSTING K61R-FmSTING K61R-RFlag-bSTING-FFlag-bSTING-RbSTING K61R-FbSTING K61R-RFlag-chSTING-FFlag-chSTING-RchSTING K67R-FchSTING K67R-RpRNF5-2HA-FpRNF5-2HA-RpRNF5-myc-FpTRIM13-myc-RpTRIM29-myc-FpRNF90-Myc-FpRNF26-myc-RpmCherry-C1-pRNF5-FpmCherry-C1-pRNF5-RpmCherry-pRNF5 1-158-RpmCherry-pRNF5 1-118-RpmCherry-pRNF5 1-68-RpmCherry-pRNF5 69-180-FhUSP20-Myc-FhUSP20-Myc-RpUSP20-myc-FpUSP20-myc-RpUSP35-myc-FpUSP44-myc-FpUSP49-myc-FsUSP49-myc-R1pUSP49-myc-R2pCYLD-myc-FpOTUD5-myc-FpEIF3F-myc-FUSP20 Δ1-111-Myc-FUSP20Δ145-685-Myc-FUSP20 Δ145-685-Myc-RUSP20 Δ687-780-Myc-FUSP20 Δ687-780-Myc-RUSP20 Δ789-914-Myc-RUSP20 Δ687-914-Myc-RpSQSTM1-Myc-FpSQSTM1-Myc-RpCCDC50-Myc-FpCCDC50-Myc-RpNPC1-Myc-FpNPC1-Myc-RpUXT-Myc-FpUXT-Myc-RpTollip-Myc-FpTollip-Myc-RpCold-GST-pSTING-FpCold-GST-pSTING-RpET32a-pUBA1-FpET32a-pUBA1-RpET32a-pUBE2D1-FpET32a-pUBE2D1-RpET32a-pRNF5-FpET32a-pRNF5-RpET32a-Ub4-K48-F1pET32a-Ub4-K48-R1pET32a-Ub4-K48-F2pET32a-Ub4-K48-R2pET32a-Ub4-K48-F3pET32a-Ub4-K48-R3 TACAAGTCCGGACTCAGATCTCCCTACTCCAGCCTGCATCCAGTACCGTCGACTGCAGAATTCTCAGAAGATATCTGAGCGGAGAAGTCCGGACTCAGATCTCCCTACTCCAGCCTGCATCCATCCGTACCGTCGACTGCAGAATTCTCAGAAGATATCTGAGCGGAGGACAAGCTTGCGGCCGCGAATTCAATGCCCTACTCCAGCCTGCCCGGGATCCTCTAGAGTCGACTCAGAAGATATCTGAGCGGAGCAGAGCCCCCTCACCAGCAGTCCTATCTGCAGATAGGACTGCTGGTGAGGGGGCTCTGCCAGGCCAGCCCTGTCTCGGATGGAGAAGTATACTTCTCCATCCGAGACAGGGCTGGCCTGCCACGTTGAAGTTCCTTCTTTCACAGATTGCAGAGACGTCTCTGCAATCTGTGAAAGAAGGAACTTCAACGTGGCCCCCGAGTACGTTCCTGTGGCGCTGATTATATAATCAGCGCCACAGGAACGTACTCGGGGGCCCGGCCCCTGATGCCAGCACGGTACCGTGCTGGCATCAGGGGCCGGGCGGCAGAAGAGTCTGGCCTGCTCGAGCGCTCGAGCAGGCCAGACTCTTCTGCCGAAGCTTGCGGCCGCGAATTCAATGACCCGCTCCAGTCTGCATCCGGGATCCTCTAGAGTCGACTCAGGAGAAATCCGTGCGGAGCAGCTGGGACAGCTGTTAAGAGGGGTCTGCGCAGACCCCTCTTAACAGCTGTCCCAGCTGAAGCTTGCGGCCGCGAATTCAATGCCATACTCCAACCTGCATCCGGGATCCTCTAGAGTCGACTCAGATGAGGTCAGTGCGGAGACTTGGACTACTGTTGAGAAACCTCTGCTGTCTGGCCAGACAGCAGAGGTTTCTCAACAGTAGTCCAAGTAAGCTTGCGGCCGCGAATTCAATGCCTCACTCCAGCCTGCATCCGGGATCCTCTAGAGTCGACTCAGAAGACATCCGAGCGGAGCAGATGGGACTGCTGATCAGGGGAATCTGCAGTCGACTGCAGATTCCCCTGATCAGCAGTCCCATCTGAAGCTTGCGGCCGCGAATTCAATGCCCCAGGACCCGTCAACCCCGGGATCCTCTAGAGTCGACTCAGGGGCAGTCACTGCGCAGGCTCGGGGTGCTGCTCAGGGGCTGCTGCAGCAGCCCCTGAGCAGCACCCCGAGCTCTCATCATTTTGGCAAAGAATTCATGGCAGCAGCGGAGGAGATCGTATGGGTAGCTGGTGATATCAATACTGAGCAGCCAGAATCTCATCATTTTGGCAAAGAATTCATGGCAGCAGCGGAGGAGGATGAGTTTCTGCTCGATATCAATACTGAGCAGCCAGAAAAACATCATTTTGGCAAAGAATTCATGGAGCTGCTGGAAGAAGATGATGAGTTTCTGCTCGATATCTAATAGTTTATATTTGCACACTCTCATCATTTTGGCAAAGAATTCATGGAAGCCGCAGACGCCGATGAGTTTCTGCTCGATATCTGGGGCCTCGTTGGACCCAATCATCATTTTGGCAAAGAATTCATGGCGGCGGTGGGGCCGCGGGATGAGTTTCTGCTCGATATCAGGCCAGATTCGCAAATAGGTCATCATTTTGGCAAAGAATTCATGGAGGCTGTGTATCTAGTAGATGAGTTTCTGCTCGATATCGAGGTAGACATTGAGGGTCTGTACAAGTCCGGACTCAGATCTATGGCAGCAGCGGAGGAGGAGGTACCGTCGACTGCAGAATTCTCAAATACTGAGCAGCCAGAAGTACCGTCGACTGCAGAATTCTCAGGCCGGGTGACCCTGTCCGTACCGTCGACTGCAGAATTCTCACCCAGTGTCGCCAAATGGGTACCGTCGACTGCAGAATTCTCATTTACACACTGGGCACTCTACAAGTCCGGACTCAGATCTGCTGGGATCAGCAGAGAAAAGCATCATTTTGGCAAAGAATTCATGGGGGACTCCAGGGACCTTGATGAGTTTCTGCTCGATATCCACGGCCCGCGTCTCGGCTTCCATCATTTTGGCAAAGAATTCATGGGGGACTCCAGGGACCCTGATGAGTTTCTGCTCGATATCCACGGCCCGCGTCTCAGCTTCCATCATTTTGGCAAAGAATTCATGGACAAGATCCTGGAGGCGGATGAGTTTCTGCTCGATATCGAAGACGAGTCTGTGGAAGTCCATCATTTTGGCAAAGAATTCATGCTAACGATGGATAAGTGCGATGAGTTTCTGCTCGATATCGCTAAGGATTTCATTAGAAGACATCATTTTGGCAAAGAATTCATGGATAGATGCAAACATGTAGATGAGTTTCTGCTCGATATCGCTTTGAGTCATTGCAGTGGAGATGAGTTTCTGCTCGATATCGGGGAAGGTCCGTGGTCTGCCCATCATTTTGGCAAAGAATTCATGAGTTCGGGCTTATGGAGCGATGAGTTTCTGCTCGATATCTTTGTACAGACTCATTGTTGGCATCATTTTGGCAAAGAATTCATGACTATTCTCCCCAAAAAGGATGAGTTTCTGCTCGATATCACTCTTGTCTGGGGGTGGGTCCATCATTTTGGCAAAGAATTCATGGCAGCACCGGCAGTACCGGATGAGTTTCTGCTCGATATCCAGGTTCACCAGCTTCTCGTTCATCATTTTGGCAAAGAATTCATGTCCCCGCCACCCTCCCACAAACCTCGAGGCCTCACGAGCGAGGAGGCCATGCGGCCGCATGGCCTCCTCGCTCGTGAGGCCTCGAGGTTTTTCTACAGGAAGAGCAGCTCCATCTGCCAGGTGGAGCTCCACCTGGCAGATGGAGCTGCTCTTCCTGTAGAAGATGAGTTTCTGCTCGATATCCTCGATCTCCACCTGGCAGATGATGAGTTTCTGCTCGATATCGCTGCTCTTCCTGTAGAAGAGCATCATTTTGGCAAAGAATTCATGGCGTCGCTCACCGTGAAGGATGAGTTTCTGCTCGATATCCAAGGGCGGTGGGTGTTTCGACATCATTTTGGCAAAGAATTCATGTCTGAAGTCAGCATCGACGATGAGTTTCTGCTCGATATCTGGCTTATAATGGAAACCTTTCATCATTTTGGCAAAGAATTCATGAGCGCGCGCGGCCCGGCCGATGAGTTTCTGCTCGATATCGAAATTGAGGAGCTGTTCTCGCATCATTTTGGCAAAGAATTCATGGCGACGCCCCCTAAACGGGATGAGTTTCTGCTCGATATCGTGGCGAGGCTCGGGGAAATTCATCATTTTGGCAAAGAATTCATGGCGACCACCGTCAGCACGGATGAGTTTCTGCTCGATATCGGACTCCTCGCCCATCTGGAGATGGAGCTCGGTACCCTCGAGATGCCCTACTCCAGCCTGCATCAGGTCGACAAGCTTGAATTCtcaGAAGATATCTGAGCGGAGGCTGATATCGGATCCGAATTCATGTCCAGCTCGCCGCTGTCCGTGGTGGTGGTGGTGCTCGAGGCGGATGGTGTATCGTACATAGCTGATATCGGATCCGAATTCATGGCGCTGAAGCGGATTCAGGTGGTGGTGGTGGTGCTCGAGCATTGCATATTTCTGAGTCCAGCTGATATCGGATCCGAATTCATGGCAGCAGCGGAGGAGGAGGTGGTGGTGGTGGTGCTCGAGAATACTGAGCAGCCAGAAAAAGCTGATATCGGATCCGAATTCATGCAGATCTTCGTGAGGACCGGTCCTCACGAAGATCTGCATTTTCCCACCTCTGAGACGGAGCTCCGTCTCAGAGGTGGGAAAATGCAGATCTTCGTGAGGACCGGTCCTCACGAAGATCTGCATCTTCCCACCTCTGAGACGGAGCTCCGTCTCAGAGGTGGGAAGATGCAGATCTTCGTGAGGACCGTGGTGGTGGTGGTGCTCGAGTCACCCACCTCTGAGACGGAG

[0055] The technical solutions of the present application will be further described in detail below in combination with examples: The present embodiment is implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following examples. Example 1

[0056] 1. Screening of the degradation pathway of activated pig STING protein First, screening was performed to determine whether double-stranded DNA mimic poly dA:dT (2 μg / mL) but not RNA mimic poly I:C (2 μg / mL) stimulation could significantly cause the degradation of activated pig STING in both pig primary alveolar macrophages (PAMs) and pig alveolar macrophage cell line 3D4 / 21 cells, and inhibit the subsequent phosphorylation of TBK1 and IRF3 (see Figure 1(A and B). Further analysis of the degradation pathway after porcine STING activation revealed that treatment with 3-MA (50 μM), NH4Cl (25 mM), and MG132 (10 μM) in 3D4 / 21 or 293T cells inhibited autophagy, lysosomes, and proteasome functions, respectively. It was found that all three treatments could, to varying degrees, reverse the degradation effects following 2'3'-cGAMP (2 μg / mL) stimulation or STING (1 μg / mL) overexpression (see A and B). Figure 1 (C and D), thus revealing that in addition to the autophagy-lysosome pathway, the ubiquitin-proteasome pathway is also essential in the negative feedback regulation of STING signaling. In 3D4 / 21 cells, stimulation with 2'3'-cGAMP or infection with HSV-1 showed that porcine STING underwent significant polyubiquitination modification after activation with prolonged stimulation or infection time (see C and D). Figure 1 (E and F). Furthermore, consistent results were obtained in porcine primary alveolar macrophages infected with ASFV, with increased STING ubiquitination levels as ASFV infection titers increased (see E and F). Figure 1 These results reveal that after DNA viral infection, STING activation primarily leads to degradation via the autophagy-lysosome and ubiquitin-proteasome pathways.

[0057] 2. Systematic screening and analysis of all 6 K sites of porcine STING mediating protein degradation and affecting signaling. STING-mediated autophagy is a primitive and conserved function that has been extensively studied in recent years. While the ubiquitin-proteasome degradation pathway of STING has been well understood in humans and mice, its definition and understanding in pigs are lacking, and reports from different species often differ. Therefore, this study aimed to investigate the mechanism mediating the ubiquitin-proteasome degradation of porcine STING. Ubiquitination modification often occurs at lysine (K) residues of target proteins. To this end, six lysine residues in porcine STING (pSTING) were mutated to arginine (KR), or five other amino acids were mutated, retaining only one lysine (KO), or all lysine residues were mutated to arginine (K0), resulting in 13 mutants of pSTING. Transfection of the constructed plasmids (500 ng / mL) into 293T cells revealed that the 61st lysine (K) mutation (KR) in pSTING significantly enhanced protein expression and increased the activity of downstream ISRE and NF-κB promoters (see [link to study].) Figure 2 In contrast, retaining only K61 and mutating the other five lysine residues in pSTING K61O allows the pSTING protein to maintain an expression level and signaling activity consistent with wild-type pSTING (see A). Figure 2B). This indicates that K61 is critical in mediating pSTING protein stability.

[0058] Further, pSTING and related mutants were complemented in STING knock-out 3D4 / 21 cells or overexpressed in 293T cells to detect the changes of downstream related signals. Quantitative PCR (qPCR) and Western blot (WB) results were consistent with the promoter results, mutation of K61 significantly increased the phosphorylation levels of TBK1 and IRF3 caused by STING activation, and enhanced downstream signal transduction (see Figure 2 C and D). By using proteasome inhibitor MG132 treatment, it was found that inhibition of proteasome significantly alleviated the abnormal enhancement of STING expression caused by K61 mutation, indicating that K61 site is closely related to proteasome-mediated degradation of pig STING (see Figure 2 Further, the anti-viral infection ability of pSTING K61 was detected, pSTING and related mutants were complemented in STING knock-out MA104 cells or HEK293T cells, and then infected with two DNA viruses, African swine fever virus (ASFV) and pseudorabies virus (PRV). The fluorescence results and flow cytometry results after ASFV infection showed that the mutation of pSTING K61 site significantly enhanced the ability of STING-mediated anti-ASFV infection and replication (see Figure 3 A-C). In addition, the copy number of PRV gD gene and virus plaque results were consistent with the ASFV infection results (see Figure 3 D and E).

[0059] 3, Polyubiquitination of pig STING protein at K61 site Since K61 is a negative regulator in the regulation of pSTING protein stability, and is closely related to proteasome activity, it is possible that polyubiquitination modification at this site mediates the degradation of STING protein. In order to explore whether ubiquitin molecules are connected at this site and determine the type of connection, 8 ubiquitin expression plasmids including wild type were used to co-transfect 293T cells with pSTING and pSTING K61R. The 8 ubiquitin expression plasmids include HA-Ub-WT, HA-Ub-K6O, HA-Ub-K11O, HA-Ub-K27O, HA-Ub-K29O, HA-Ub-K33O, HA-Ub-K48O, HA-Ub-K63O. The results showed that pSTING can be polyubiquitinated with multiple types of connections, however, mutation of K61 can significantly inhibit the polyubiquitination of pSTING protein at K27, K29, K33 and K48 sites (see Figure 4(A). K48-linked polyubiquitination is a crucial prerequisite for proteasome degradation; therefore, the effects of other lysine residues in porcine STING on K48-linked polyubiquitination were further investigated. It was found that a single mutation at K61 in porcine STING almost completely deactivated all K48-linked polyubiquitinations in STING (see A). Figure 4 (B). Further identification of the polyubiquitination type at this site was performed using pSTING K61O and K0 mutants. The results showed that the K61 mutation almost completely de-ubiquitinated all K48-linked pSTING molecules (see B). Figure 4 (C). Porcine E1 activator UBA1, E2 conjugate UBE2D1, Ub-K48, and GST-tagged STING and K61R were purified by expression in *E. coli* (see [link to article]). Figure 4 (D) It was found that both co-incubation of the purified protein with lysates of 293T cells expressing ubiquitin molecules and in vitro ubiquitination assays showed that the K61 site of STING is a necessary site for K48-linked ubiquitination modification (see D). Figure 4 (E and F).

[0060] 4. Ubiquitination of porcine STING at K61 regulates protein stability in a species-specific manner. The K61 site is crucial for the stability of porcine STING proteins. Therefore, a comparative analysis was conducted on the conservation of STING at this site in nine species, including humans, African green monkeys, mice, pigs, cattle, and birds. The results showed that, except for humans, other mammals, and even birds, showed high conservation at this homologous site (see...). Figure 5 Therefore, it is reasonable to speculate whether the STING K61 homologous site in other species also has a similar function. STING and corresponding K61 homologous site mutants from monkeys, mice, cattle, and chickens were cloned and transfected into 293T cells to detect related signals. The results were contrary to expectations; except for porcine STING, mutations at the K61 homologous site in STING from other species did not affect its stability or enhance subsequent TBK1 and IRF3 phosphorylation (see A). Figure 5 (B). The results of dual-luciferase reporter assay and qRT-PCR were consistent with the results of Western blot, except that the porcine STING K61 mutation significantly enhanced downstream type I interferon (IFN) signaling (see B). Figure 5 (C and D). Further ubiquitination modification was identified in STING K61 homologous mutants from the above species. The results showed that only the deletion mutation in porcine STING K61 significantly reduced the levels of total ubiquitination and K48-linked ubiquitination (see C and D). Figure 5 The E), while mutations at the STING K61 homologous site in monkeys, mice, cattle, and chickens did not affect the ubiquitin modification of the protein itself (see E). Figure 5Figure 6. Ubiquitination of pig STING K61 is species-specific. (A) Schematic diagram of pig STING K61 ubiquitination. (B) Ubiquitination of pig STING K61 by RNF5. (C) Ubiquitination of pig STING K61 by RNF5 in the presence of ATP. (D) Ubiquitination of pig STING K61 by RNF5 in the presence of ATP and ubiquitin aldehyde. (E) Ubiquitination of pig STING K61 by RNF5 in the presence of ATP and ubiquitin aldehyde. (F) Ubiquitination of pig STING K61 by RNF5 in the presence of ATP and ubiquitin aldehyde. These results indicate that ubiquitination of pig STING K61 is species-specific.

[0061] Unlike other species of mammals, including pigs, cattle, sheep, STING of various domestic animals has fewer lysine (K) residues, which suggests that there may be species differences. While the homology between bovine STING and pig STING is 87.3%, which is significantly higher than the homology between pig STING and other non-domestic animal STING Figure 4 A), so it is speculated that the difference in the amino acids adjacent to STING K61 of pig and bovine species may affect the binding of ubiquitin molecules. By comparing the amino acid differences between the two STINGs on both sides of K61, the I60 and I63 of bovine STING were mutated to V60 and L63, which are the same as pig STING, to obtain bovine STING I60V I63L mutant, which is named M1, and the same mutation was made to bovine STING K61R to obtain bovine STING I60V K61R I63L mutant, which is named M2 (see Figure 5 Figure 7. Ubiquitination of bovine STING K61 is affected by mutations in the amino acids adjacent to K61. (A) Schematic diagram of bovine STING K61 ubiquitination. (B) Ubiquitination of bovine STING K61 by RNF5. (C) Ubiquitination of bovine STING K61 by RNF5 in the presence of ATP. (D) Ubiquitination of bovine STING K61 by RNF5 in the presence of ATP and ubiquitin aldehyde. (E) Ubiquitination of bovine STING K61 by RNF5 in the presence of ATP and ubiquitin aldehyde. (F) Ubiquitination of bovine STING K61 by RNF5 in the presence of ATP and ubiquitin aldehyde. These results indicate that ubiquitination of bovine STING K61 is affected by mutations in the amino acids adjacent to K61. Figure 5

[0062] 5、RNF5 as E3 ligase enhances K29-, K33- and K48-linked polyubiquitination of pig STING Ubiquitin molecules require three ubiquitin-related proteases to be involved in the process of binding to target proteins and modifying them, among which the E3 ubiquitin ligase transfers the activated ubiquitin molecule from the E2 binding enzyme to the substrate protein, thereby achieving ubiquitination labeling of the target protein. Unlike the relatively small number of E1 activase and E2 binding enzymes, there are hundreds of E3 ubiquitin ligases in mammals. In order to find out the E3 ubiquitin ligase that mediates the polyubiquitination of pSTING K61 site, a plurality of E3 ubiquitin ligases reported to mediate STING degradation in humans and mice were cloned on pigs. Including TRIM29, TRIM13, RNF90 and RNF5, among which TRIM29, RNF90 and RNF5 enhance the ubiquitin-proteasome degradation pathway of STING by promoting the K48-linked polyubiquitination of STING, and TRIM13 catalyzes the Lys6-linked polyubiquitination of STING, which slows down the transport speed of STING from ER and accelerates the ER-induced degradation of STING.

[0063] ​Co-expression of these E3 ligases with STING protein in 293T cells, we found that only RNF5 co-expression with pSTING can significantly reduce the expression of pSTING protein (see Fig. 1A). Further analysis of the mechanism, we found that all the other four E3 ubiquitin ligases have obvious interaction with STING and cytoplasmic co-localization, but only RNF5 can significantly enhance the total ubiquitination of pSTING and K48-linked polyubiquitination (see Fig. 1B-D). Immunoprecipitation of endogenous STING in 3D3 / 21 cells, HSV-1 infection significantly enhanced the interaction of STING and RNF5 (see Fig. 2E), which has the same trend as the ubiquitination level of STING after DNA virus infection. In addition, RNF5 promotes the degradation of pSTING in a dose-dependent manner and inhibits various signals such as STING downstream interferon (IFN) response and autophagy (see Fig. 3A-C). While the mutation of K61 can significantly reverse the degradation of pSTING by RNF5 (see Fig. 4D). By expressing and purifying RNF5 protein in E. coli (see Fig. 5E), GST pulldown experiment showed that RNF5 can interact with STING in vitro (see Fig. 6F), and modify STING with K48-linked polyubiquitination (see Fig. 7G). Not only that, RNF5 also significantly enhances the K27, K33 and K48-linked polyubiquitination of pSTING (see Fig. 8H). When only the lysine at position 61 of STING is retained, RNF5 can significantly enhance the K48-linked polyubiquitination of STING, without affecting other types of ubiquitination (see Fig. 9I). On the contrary, when only K61 (K61R) is mutated, RNF5 significantly enhances the K29 and K33-linked polyubiquitination of pSTING, without affecting the K48-linked polyubiquitination (see Fig. 10J). These results show that K61 is a key site for mediating K48-linked polyubiquitination of porcine STING, and RNF5 specifically enhances K48-linked polyubiquitination at K61 site. Figure 6 Figure 6 Figure 6 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7

[0064] 6、CBD region of porcine STING protein and TM of RNF5 are the key regions for interaction ​​​​​​​​​​​To determine the key domain of RNF5 interacting with STING and promoting ubiquitination, the key domains of STING and RNF5 were analyzed by online website Uniprot and were truncated. The truncation body composition is shown in the schematic diagram. The domain of STING protein can be divided into the transmembrane domain at the N terminal, the dinucleotide binding domain in the middle and the terminal tail region at the C terminal, which is the key region for regulating type I interferon (IFN) signal. By retaining or deleting different domains separately, 8 STING-related truncated / deleted mutants were obtained (see Figure 8 A). The 238-241 amino acids of STING were reported to be the key amino acid motif for mediating autophagy, and therefore a deletion body lacking this region was also constructed. The positive and negative immunoprecipitation results showed that although the deletion of the transmembrane domain of STING severely inhibited its interaction with RNF5, it was not an essential domain for the interaction of porcine STING with RNF5. The presence of the CBD domain alone was sufficient to produce strong interaction with RNF5, and the deletion of this region completely inhibited this interaction (see Figure 8 B and C), which was different from the previous report in human species. The intracellular co-localization results also further confirmed the results, and the deletion of the CBD region of porcine STING significantly changed the subcellular localization of the STING protein and significantly reduced the co-localization phenomenon with RNF5 (see Figure 8 D).

[0065] RNF5 is a common E3 ubiquitin ligase with a ring finger domain, which is a small molecular weight protein with simple structure. In addition to the ring finger domain, it also has an undefined function domain and two transmembrane regions (see Figure 8 E). RNF5 was deleted in sections, and the change in its interaction with STING was detected, and the positive and negative immunoprecipitation results showed that the deletion of TM2 of RNF5 alone significantly inhibited its interaction with STING, while the deletion of the RING domain had no effect on its interaction with STING (see Figure 8 F and G). Immunofluorescence further analyzed that TM2 of RNF5 is the key sequence for its passage through the nuclear membrane. The complete RNF5 is a completely cytoplasmic expression protein, and has strong co-localization with STING. However, the deletion of TM2 changes the subcellular localization of RNF5, causing it to be mostly expressed in the nucleus, significantly inhibiting its co-localization with STING (see Figure 8 H).

[0066] 7、RNF5 negatively regulates the anti-viral response mediated by porcine STING To further analyze the regulatory effect of RNF5 on STING protein and downstream signal, RNF5 was knocked out in 3D4 / 21 cells by CRISPR-cas9, and homozygous RNF5 knockout was obtained. Two homozygous clones were selected to detect their response to STING agonists. Reverse transcription quantitative PCR (RT-qPCR) results showed that RNF5 knockout enhanced the transcription of IFN-β and related interferon-stimulating genes ISG after STING activation (see Fig. A of Figure 9 From a mechanism point of view, porcine RNF5 knockout significantly enhanced STING protein expression, delayed STING degradation after activation, and enhanced downstream TBK1 and IRF3 phosphorylation (see Fig. B of Figure 9 , thereby enhancing STING-mediated type I interferon (IFN) response. At the same time of RNF5 deletion, treatment with autophagy and lysosome inhibitors 3-MA and NH4Cl showed that simultaneous inhibition of ubiquitin proteasome pathway and autophagy lysosome pathway could most effectively alleviate the degradation effect after STING activation, and enhance downstream signal transduction (see Fig. C and D of Figure 9 Further exploration of STING ubiquitination modification after RNF5 knockout was similar to the results in 293T cells, although not completely inhibited, RNF5 knockout also significantly reduced the ubiquitination level of STING after HSV-1 infection (see Fig. E of Figure 9 In RNF5 knockout 3D4 / 21 cells, complementation of RNF5 and TM2 deletion mutants showed that double luciferase reporter assay and real-time fluorescence quantitative PCR results showed that complementation of RNF5 significantly reduced the expression of interferon-stimulating genes and inflammatory genes mediated by STING activation (see Fig. F and G of Figure 9 From a mechanism point of view, complementation of RNF5 further enhanced the degradation of STING mediated by STING agonist treatment, while deletion of RNF5 TM2 eliminated this effect and restored the downstream multiple signal transduction activated by STING activation (see Fig. H of Figure 9 Detection of porcine RNF5 regulation of cGAS-STING-mediated antiviral signal showed that whether PRV, HSV-1 or VSV infection, deletion of RNF5 enhanced the cell's basic broad-spectrum antiviral ability, and also improved the antiviral signal caused by STING activation (see Fig. I and J of Figure 9 The viral replication level and viral titer of RNF5 knockout cells were significantly lower than those of wild type control cells.

[0067] 8、USP20 is the main deubiquitinase for removing K48-linked polyubiquitin chains from porcine STING K61 site Post-translational modifications of proteins often have dual regulatory effects, such as protein kinases that mediate phosphorylation and their corresponding phosphatases working together to maintain homeostasis of protein phosphorylation, thereby balancing intracellular function. Unlike other post-translational modifications, proteins labeled with ubiquitin chains mostly follow three pathways: first, they are recognized and degraded by the 26S proteasome; second, they are recognized by autophagy receptors and transported to autolysosomes for degradation; and third, after performing specific functions, the ubiquitin chain is removed by deubiquitinases for cellular recycling. Compared to E3 ligases, STING has a wide variety of deubiquitinases, many of which are members of the ubiquitin-specific protease family, including USP20, USP44, and USP49. In pigs, combined with reports from humans and mice, eight proteases that can directly or indirectly affect STING ubiquitination were cloned and constructed. The results showed that multiple proteins, including USP20, USP35, CYLD, EIF3F, and RNF26, significantly removed STING ubiquitination, while USP20 and RNF26 almost completely removed RNF5-mediated polyubiquitination of STING (see...). Figure 10 Both negatively regulate STING post-translational modifications through different mechanisms. RNF26 exerts its inhibitory effect on STING ubiquitination modification by reducing RNF5 expression and inhibiting its interaction with STING (see A). Figure 10 (B), while USP20 can directly remove the polyubiquitin chain linked at the K48 position of STING, whether RNF5 is present or not.

[0068] Furthermore, USP20 knockout 3D4 / 21 cells were constructed. Contrary to the results of RNF5 deletion, USP20 deletion significantly inhibited multiple signaling pathways following STING agonist stimulation, including phosphorylation of TBK1 and IRF3, and lipidation of LC3 (see [link to study]. Figure 11 The A-type IFN-β and ISG transcriptional type I interferon responses were also reduced to varying degrees. Figure 11 B). Mechanistically, the deletion of USP20 enhances the ubiquitination and subsequent degradation of STING following HSV-1 infection (see [link to article]). Figure 11 The absence of B and C in STING (USP20) further affects multiple signaling pathways mediated by STING. Furthermore, the absence of USP20 also reduces the broad-spectrum antiviral effects of STING, including those against HSV-1, VSV, and PRV (see [link to relevant documentation]). Figure 11 DH).

[0069] The application first identifies the species specificity key site K61 of the pig STING protein, which is unique to pigs and different from other species, and plays a key negative regulation role in the replication of viruses such as ASFV, PRV, HSV-1, etc. through regulating protein stability and downstream signal transduction; at the same time, the K61 site can be directly used as a core molecular marker and a targeted site for pig antiviral breeding, providing important support for improving the efficiency of antiviral breeding research.

[0070] The above is only a specific implementation in the application, but the protection scope of the application is not limited thereto, any person skilled in the art can understand and think of changes or replacements within the technical range disclosed by the application, which should be covered in the inclusive scope of the application, therefore, the protection scope of the application should be subject to the protection scope of the claims.

Claims

1. A porcine STING protein K61R mutant, characterized in that, The mutant is a mutation of lysine at position 61 of the porcine STING protein to arginine, and the amino acid sequence of the porcine STING protein is shown in SEQ ID NO.

1.

2. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the porcine STING protein K61R mutant as described in claim 1.

3. A carrier, characterized in that, The carrier comprises the polynucleotide molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector of claim 3 or the host cell's chromosome integrates the polynucleotide molecule of claim 2.

5. The application of the porcine STING protein K61R mutant as described in claim 1, the polynucleotide molecule as described in claim 2, the vector as described in claim 3, or the host cell as described in claim 4 in broad-spectrum antiviral infection.

6. The application according to claim 5, characterized in that, The use of the porcine STING protein K61R mutant, polynucleotide molecule, vector, or host cell in the preparation of drugs for the prevention and / or treatment of swine viral infections.

7. The application according to claim 6, characterized in that, The swine viruses mentioned include, but are not limited to, African swine fever virus, pseudorabies virus, vesicular stomatitis virus, and herpes simplex virus-1.

8. The application of the porcine STING protein K61R mutant as described in claim 1, the polynucleotide molecule as described in claim 2, the vector as described in claim 3, or the host cell as described in claim 4 in the breeding of antiviral pigs.

Images