Application of porcine I-type interferon receptor IFNAR2 protein mutant in resisting African swine fever virus

Abstract

The invention discloses a pig IFNAR2 protein mutant, which is characterized in that the 399th site in the amino acid sequence of the pig IFNAR2 protein is mutated from glycine to alanine, and the amino acid sequence of the pig IFNAR2 protein is as shown in SEQ ID NO. 1. The invention provides a specific cleavage site of ASFV protease pS273R, and the site is mutated through a gene editing technology so as to block the cleavage of pig IFNAR2 by the pS273R and improve the ability of pigs to resist African swine fever virus infection. By blocking ASFV from damaging type I IFN mediated JAK-STAT signal activation, on the basis of retaining pig autoimmune performance, higher specificity and higher efficiency are achieved, negative effects of genetic engineering on pig growth performance and reproductive performance can be greatly reduced, and biosafety is higher.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the porcine type I IFN receptor IFNAR2 protein mutant in the fight against African swine fever virus. Background Technology

[0002] African swine fever (ASF) is a highly virulent and fatal infectious disease of domestic and wild pigs caused by African swine fever virus (ASFV). ASFV is the only member of the African swine fever virus family and is a large, enveloped, double-stranded DNA virus transmitted by soft ticks. ASFV has a complex structure, resembling an icosahedron, and consists of five parts from the outside in: the outer membrane, capsid, inner membrane, core capsid, and genome. The viral genome is large, approximately 170-192 kb, encoding over 150 proteins, including 68 structural proteins and over 100 non-structural proteins.

[0003] The ASFV protease pS273R, encoded by the S273R gene in the viral genome, is approximately 31 kDa in size. This protein contains two domains: an N-terminal "Arm domain (AD)" and a C-terminal "Core domain (CD)". pS273R belongs to the SUMO-1 specific protease family. Similar to typical SUMO proteases, its C-terminal active site consists of a catalytic triplet (Cys232, His168, Asp187) that precisely cleaves the ASFV polyprotein precursors pp220 and pp62 at the X-Gly-Gly-XX motif (where X is any amino acid), producing mature viral structural proteins. Specifically, pp220 is cleaved by pS273R to produce p150, p40, p37, and p34, while pp62 is cleaved to produce p35 and p15. This process is crucial for the correct assembly of the viral core capsid. Inhibiting or mutating the activity of pS273R leads to incomplete processing of the polyprotein, preventing the formation of infectious mature viral particles, thereby effectively blocking viral replication.

[0004] Innate immunity, also known as natural immunity, is the body's first line of defense against invading pathogens. Pattern recognition receptors (PRRs) encoded by germline genes rapidly initiate inflammatory and anti-infective responses by recognizing conserved pathogen-associated molecular patterns (PAMPs) common to pathogens and their own damage-associated molecular patterns (DAMPs), providing the environmental basis for subsequent adaptive immunity. Innate immune pattern molecular recognition receptors include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin-like receptors (CLRs), and the cytoplasmic DNA receptor (CDR) family. The PAMPs they recognize include nucleic acids, lipopolysaccharides, flagellins, etc. Among various PAMPs, nucleic acids are the main PAMPs in viral infection; therefore, RIG-I-like receptors and cytoplasmic DNA receptors that sense nucleic acids play a crucial role in cellular antiviral responses. During viral infection, pattern recognition receptors (PRRs) detect the pathogen and recognize the corresponding PAMPs (e.g., RLR recognizes viral RNA, or cGAS recognizes viral DNA). They then transmit the signal to downstream adaptor proteins STING / MAVS. These adaptor proteins recruit relevant kinases (e.g., the TBK1 and IKK complex) to activate transcription factors (e.g., IRF3 / 7), inducing the synthesis of type I interferon (IFN-α / β) and type III interferon (IFN-λ), as well as the expression of a small number of interferon-stimulated genes (ISGs). The synthesized interferon (IFN), acting as a signaling molecule, acts on the cell itself and surrounding cells via autocrine or paracrine pathways. It binds to interferon receptors (IFNAR1 / IFNAR2 and IFNLR1 / IL10R2) on the cell surface. This binding increases the affinity of the previously loose "receptor dimer," forming a stable receptor complex. Kinases JAK1 and TYK2, which bind to the intracellular region of the receptor, also approach each other and undergo transphosphorylation and activation. Signal transducers and activators of transcription 1 (STAT1) and STAT2 are phosphorylated by activated protein kinases to form heterodimers, which recruit IRF9 to form the ISGF3 ternary complex. After ISGF3 is transported into the nucleus, it binds to interferon-stimulated response elements (ISREs) on target genes, rapidly inducing the expression of hundreds of interferon-stimulated genes (ISGs). These gene products collectively establish an "antiviral state" network capable of inhibiting viral replication and spread at multiple stages.

[0005] ASFV possesses a complex immune evasion strategy, encoding multiple proteins to antagonize the host's innate immune response through various pathways. These include: reducing type I IFN secretion by inhibiting the DNA receptor cGAS-STING signaling pathway and the RNA receptor RIG-I-MAVS signaling pathway; targeting the JAK-STAT pathway to inhibit the production of antiviral ISGs; and attenuating the inflammatory response following ASFV infection by inhibiting the NF-κB pathway and the production of the NLRP3 inflammasome. These immune evasion mechanisms collectively constitute ASFV's complex defense system, with interference with the IFN pathway and regulation of the inflammatory response being particularly crucial. Currently, the development strategy for gene deletion vaccines against ASFV mainly involves obtaining attenuated strains by deleting genes related to viral virulence / pathogenicity, with genes exhibiting immune evasion often being the preferred targets.

[0006] pS273R is a key protein in ASFV immune escape, and various immune escape mechanisms have been reported. Based on its protease properties, pS273R antagonizes the host's innate immune signals and can be divided into two categories: SUMO-dependent and SUMO-independent. Regarding immune escape based on its enzymatic activity, pS273R can: 1) cleave the host's GSDMD to inhibit pyroptosis and cellular inflammatory responses; 2) cleave G3BP1 to inhibit the formation of stress granules; and 3) inhibit cGAS-STING-mediated type I IFN production by removing the SUMOylation of IKKe. Regarding immune evasion independent of its enzymatic activity, pS273R inhibits IFN signaling by interfering with the interaction between TBK1 and IRF3 and reducing the expression of the antiviral factor FoxJ1. In addition, pS273R can bind to STAT2 and recruit the E3 ligase DCST1 to promote the degradation of STAT2, thereby inhibiting the production of antiviral ISG stimulated by IFN. pS273R can also inhibit NF-κB signaling and promote viral replication by blocking the translocation of IκBα to the proteasome.

[0007] Although the mechanism of pS273R in immune evasion has been extensively studied, most research has focused on the upstream of the IFN response, namely IFN production. However, the body's antiviral immune response strongly depends on IFN stimulation-induced high expression of ISG. Research on the IFN-JAK-STAT signaling axis is limited. Current research has screened and discovered that the African swine fever virus protease pS273R precisely cleaves the interferon receptor protein IFNAR2, elucidating how the virus blocks the association between IFN and ISG during ASFV infection. Therefore, breeding and screening for IFNAR2 gene mutants resistant to pS273R cleavage is a potential direction for ASFV-resistant breeding. Summary of the Invention

[0008] This invention addresses the technical problem at hand and overcomes the shortcomings of existing technologies by providing an application of a porcine type I IFN receptor IFNAR2 protein mutant in the fight against African swine fever virus.

[0009] This invention reveals that during ASFV infection, porcine IFNAR2 is cleaved at site G399 by the ASFV protease pS273R, disrupting the molecular structure of porcine (p)IFNAR2, weakening its interaction with IFNAR1 and JAK1, and significantly inhibiting the activation of type I IFN-mediated JAK-STAT signaling and the production of antiviral ISG. By targeting the cleavage site of pIFNAR2 and editing the porcine IFNAR2 gene and / or other related genes using gene editing technology, breeding pigs resistant to African swine fever can be bred. Therefore, the discovery of protease cleavage of porcine IFNAR2 provides an important key target for genetic breeding of ASF-resistant pigs and offers a new approach to controlling porcine viral infections.

[0010] In a first aspect, the present invention provides a porcine IFNAR2 protein mutant, namely G399A, wherein the mutant is formed by changing glycine (Gly) to alanine (Ala) at position 399 of the amino acid sequence of the porcine IFNAR2 protein, and the amino acid sequence of the porcine IFNAR2 protein is shown in SEQ ID NO.1.

[0011] This invention provides a new target for the breeding or preparation of antiviral pigs, which is of great significance for improving the level of disease-resistant breeding research.

[0012] In a second aspect, the present invention provides a polynucleotide molecule encoding the porcine IFNAR2 protein mutant.

[0013] Thirdly, the present invention provides a carrier comprising the polynucleotide molecule.

[0014] Fourthly, the present invention provides a host cell that contains the vector or chromosome integrating the polynucleotide molecule.

[0015] Fifthly, the present invention provides the application of the porcine IFNAR2 protein mutant, the polynucleotide molecule, the vector, or the host cell in the fight against African swine fever virus infection.

[0016] In the above application, by site-directed mutation of the amino acid at position 399 of the porcine IFNAR2 protein, changing it from glycine to alanine, and then reintroducing it into porcine alveolar macrophage cell lines with the IFNAR2 gene knocked out, the replication of African swine fever virus in infected cells was inhibited.

[0017] In a sixth aspect, the present invention provides the use of the porcine IFNAR2 protein mutant, the polynucleotide molecule, the vector, or the host cell in the preparation of a medicament for the prevention and / or treatment of African swine fever virus infection.

[0018] Seventhly, this invention provides the application of the porcine IFNAR2 protein mutant, the polynucleotide molecule, the vector, or the host cell in breeding antiviral pigs. This invention provides a specific cleavage site for the ASFV protease pS273R and uses gene editing technology to mutate this site, thereby blocking the cleavage of porcine (p)IFNAR2 by pS273R, thus improving the pig's resistance to African swine fever virus infection. Compared to traditional antiviral drugs and vaccines, this invention, by blocking ASFV's disruption of type I IFN-mediated JAK-STAT signal activation, exhibits stronger specificity and higher efficiency while preserving the pig's own immune function. It can significantly reduce the negative impact of genetic engineering on pig growth and reproductive performance, and has higher biosafety. Attached Figure Description

[0019] Figure 1 This is a screening diagram of the effects of ASFV protease pS273R on DNA and RNA receptor signaling pathways. In the diagram, A and B represent 3D4 / 21 cells stably expressing pS273R treated with various DNA and RNA receptor agonists and control cells, respectively. Luciferase reporter assays were used to detect differences in downstream IFN-β and ISRE promoter activities, and qRT-PCR was used to detect the expression of related downstream genes. C represents 3D4 / 21 cells stably expressing pS273R stimulated with polydA:dT and poly I:C and control cells, with Western blotting used to detect the phosphorylation levels of downstream TBK1, IRF3, and STAT1.

[0020] Figure 2 This is a screening diagram showing the effect of ASFV protease pS273R on the activation of the JAK-STAT pathway by type I and type III IFN (IFN-I / III). Figures A and B show the results of a luciferase reporter assay detecting pS273R transfection with IRF3. - / - The effects of 3D4 / 21 cells on the activity of the ISRE promoter activated by IFN-I and IFN-III and the changes in the expression of multiple downstream ISGs detected by qPCR. C and D are the results of Western blotting detection of pS273R transfection with IRF3. - / -The effect of 3D4 / 21 cells on IFN-α and IFN-β activated STAT1 phosphorylation: E shows the effect of pS273R transfection of PK15 cells on IFN-I and IFN-III activated ISG15 and CXCL10 transcription by qRT-PCR; F shows the difference in expression of various downstream ISGs detected by qPCR between IFN-I and IFN-III treatment of 3D4 / 21 cells stably expressing pS273R and control cells.

[0021] Figure 3 This diagram illustrates the interactions and cleavage between pS273R and IFN receptors, downstream kinases, and transcription factors. Figure A shows co-transfection of pS273R with GFP-tagged IFNAR1, IFNAR2, IFNLR1, IL-10R2, and IRF9 in HEK293T cells, followed by immunoprecipitation of pS273R and Western blotting to detect the interaction between these proteins and pS273R. Figure B shows co-transfection of pS273R with HA-tagged JAK1, TYK2, STAT1, and STAT2 in HEK293T cells, followed by immunoprecipitation of pS273R and Western blotting to detect the interaction between these proteins and pS273R. Figures C and D show the same transfection treatment as figures A and B in 3D4 / 21 cells, followed by immunofluorescence staining and laser confocal microscopy to observe the co-localization of pS273R with these proteins. Figures E and F show the same transfection treatment as figures A and B in HEK293T cells, followed by Western blotting to observe the co-localization of pS273R with these proteins. Blotting was used to detect the cleavage of key proteins in the JAK-STAT pathway by pS273R.

[0022] Figure 4 Analytical plot of the effect of pS273R cleavage of IFNAR2 on downstream signal transduction. Figure A shows the effect of different concentrations of pS273R on IFNAR2 cleavage in pigs as detected by Western blotting; Figure B shows the effect of different concentrations of pS273R on IFNAR2 cleavage in IRF3. - / - 3D4 / 21 cells were infected with different doses of ASFV, and Western blotting was used to detect the effect of ASFV infection on endogenous IFNAR2 protein cleavage; C is W, which is the effect of pS273R on exogenous IFNAR2 expression and IFNAR2 interaction detected by immunoprecipitation; D is the effect of pS273R on IFNAR2-JAK1 interaction detected by immunoprecipitation.

[0023] Figure 5The figure shows the effect of pS273R enzyme activity on the cleavage of pIFNAR2. Figure A is a schematic diagram of the construction of pS273R protease active site mutants and truncated variants; B is the Western blotting detection of the cleavage activity of the two truncated variants of pS273R on IFNAR2 protein; C is the Western blotting detection of the cleavage activity of the three enzyme active site mutants of pS273R on IFNAR2 protein; D is the co-localization of the three enzyme active site mutants of pS273R with IFNAR2 observed by laser confocal microscopy.

[0024] Figure 6 Figure 1 shows the construction and functional identification results of IFNAR2 knockout 3D4 / 21 cells. In the figure, A represents the constructed IFNAR2 cells. - / - Genotypic comparison of 3D4 / 21 cells and parental WT cells; B and C represent the two constructed IRF3 strains, respectively. - / - IFNAR2 - / - 3D4 / 21 cells and parental IRF3 - / - Genotype comparison of 3D4 / 21 cells; D shows the effect of qRT-PCR on the expression of IFNAR2 knockout on the expression of IFN-β, TNF-α, and ISG15 genes activated by poly I:C and poly dA:dT stimulation; E and F show the effects of qRT-PCR on IFNAR2 knockout in 3D4 / 21 cells or IRF3 cells, respectively. - / - Effects of IFN-I and IFN-III stimulation on the expression of ISG genes activated in 3D4 / 21 cells.

[0025] Figure 7 Figure 1 shows the results of the role of IFNAR2 in anti-ASFV infection. Figure A shows the effect of IFNAR2 knockout on the ASFV infection effect in 3D4 / 21 cells as observed by fluorescence microscopy and flow cytometry; B is a statistical graph of the flow cytometry results in Figure A; C is the effect of IFNAR2 knockout on the expression of p72 and p30 proteins after ASFV infection in 3D4 / 21 cells as detected by Western blotting; D is the effect of IFNAR2 knockout on the IFN-I stimulation-mediated anti-ASFV infection effect in 3D4 / 21 cells as observed by fluorescence microscopy and flow cytometry; E is a statistical graph of the flow cytometry results in Figure D; F shows the difference in p72 and p30 protein expression after ASFV infection as detected by Western blotting under the same conditions as Figure D.

[0026] Figure 8Figure 1 shows the site identification results of pIFNAR2 protein cleavage by the protease pS273R. Figure A shows the cleavage sites of ASFV precursor proteins pp62 and pp220 by pS273R and potential cleavage sites of pIFNAR2; Figure B is a schematic diagram of the construction of four potential cleavage site mutants of pIFNAR2; Figure C shows the differences in pIFNAR2 and the four mutants cleaved by pS273R as detected by Western blotting; Figures D and E show the interaction between pIFNAR2 and the four mutants with pS273R as detected by Western blotting and the co-localization changes observed by laser confocal microscopy, respectively; Figure F shows the interaction between IFNAR2 protein after the G399 mutation and JAK1 in the presence of pS273R, as detected by Western blotting; Figure G shows the interaction between IFNAR2 and JAK1 in the presence of pS273R. - / - 3D4 / 21 cells were reintroduced with IFNAR2 and four mutants, and qRT-PCR was used to detect the effect of the presence or absence of pS273R on the expression of multiple ISGs activated by IFN-I.

[0027] Figure 9 The figure shows the results of the analysis of the interaction regions of pS273R and IFNAR2. In the figure, A represents the domains of pS273R and pIFNAR2 interaction detected by immunoprecipitation; B represents the domains of pIFNAR2 and pS273R interaction detected by immunoprecipitation; and C represents the domains of pIFNAR2 interaction. - / - 3D4 / 21 cells were replenished with IFNAR2 and two cleaved fragments. qRT-PCR was used to detect whether the two cleaved IFNAR2 fragments still possessed IFN-I-mediated ISG stimulation activity.

[0028] Figure 10 Figure 1 shows the results of restoring the effects of porcine IFNAR2 and its cleavage site mutants on ASFV replication. Figure A represents the effect of IFNAR2 on ASFV replication. - / - 3D4 / 21 cells were reintroduced with IFNAR2 and four mutants and two truncated variants. Fluorescence microscopy and flow cytometry were used to observe and detect the changes in IFN-I-mediated resistance to ASFV infection in IFNAR2 mutants and truncated variants. B is a statistical graph of the flow cytometry results in Figure A. C shows the difference in p72 and p30 protein expression in 3D4 / 21 cells after ASFV infection under the same treatment as in Figure A, as detected by Western blotting. Detailed Implementation

[0029] This invention provides a porcine IFNAR2 protein mutant with a mutation at amino acid residue 399, specifically replacing glycine (Gly, G) with arginine (Ala, A). The study found that during ASFV infection, the viral protease pS273R specifically cleaves the porcine IFNAR2 protein at site G399, a cleavage strictly dependent on the integrity of the pS273R protein structure and the enzyme's active site. Cleavage of IFNAR2 by pS273R reduces its binding to IFNAR1 and JAK1, and further inhibits STAT1 phosphorylation and subsequent antiviral ISG production. Furthermore, it further confirms that IFNAR2 is an important antiviral protein during ASFV infection. IFNAR2 knockout significantly enhances ASFV infection efficiency in porcine alveolar macrophage cell lines. The translocation complementation of the IFNAR2 G399A mutant effectively antagonizes the cleavage effect of ASFV pS273R, enhancing the IFN-I-mediated antiviral effect. By targeting the cleavage site on IFNAR2 and editing the porcine IFNAR2 gene and / or related genes using gene editing technology, it is hoped that breeding pigs resistant to ASFV infection can be bred. Therefore, the discovery of the pS273R cleavage site on IFNAR2 provides an important key target for genetic breeding of pigs resistant to ASFV and offers a new approach to controlling porcine viral infections.

[0030] Materials description: (1) Plasmids, cell lines and strains

[0031] Plasmids: Eukaryotic expression plasmids pEGFP-C1, pmCherry-C1, pmCherry-C1-pS273R, p3xFlag-CMV-7.1, p3xFlag-CMV-7.1-pS273R, pCAGGS-2HA, pCAGGS-Myc, pX458-GFP, and mammalian IFN-β firefly luciferase reporter gene plasmids (IFN-β-Fluc), ISRE firefly luciferase reporter gene plasmids (ISRE-Fluc), and β-actin renin luciferase reporter plasmids (β-actin-Rluc) were all preserved by the Animal Infection and Immunology Laboratory of Yangzhou University. Among them, the empty vector plasmids pEGFP-C1 (P0134), pmCherry-C1 (P0151), p3xFlag-CMV-7.1 (P0438), and pCAGGS (P0165) were purchased from Miaoling Biotechnology. For pS273R from two different vectors, the nucleotide sequence of ASFV S273R was introduced into Benchling, and primers for amplification of different vectors were designed. The primer sequences are shown in Table S1. PCR amplification was performed using the gold-labeled MIX (green) enzyme with ASFV genomic DNA as a template, and the amplified products were recovered from the gel. The eukaryotic expression vectors pmCherry-C1 and p3xFlag-CMV-7.1 were double-digested with restriction endonucleases Bgl II / EcoRI and EcoRI / Sal I, respectively. The digested products were recovered from the gel, and the recovered target fragments were seamlessly cloned with the corresponding double-digested linear vectors using 2×MultiF Seamless Assembly Mix. pX458-GFP and luciferase reporter plasmids were purchased from Ubisoft Biotechnology.

[0032] For other expression plasmids, total RNA was extracted from 3D4 / 21 cells using TRIPURE reagent, and open reading frames (ORFs) of the following genes were obtained by RT-PCR: porcine IFNAR1 (NM_213772), porcine IFNAR2 (NM_001204775), porcine IFNLR1 (OQ947873), porcine IL-10R2 (NM_213771), porcine JAK1 (NM_001434996), porcine TYK2 (NM_001114670), porcine STAT1 (NM_213769), porcine STAT2 (NM_213889), and porcine IRF9 (NM_001078670). The primer sequences used are shown in Table S1. PCR products were cloned into the Bgl II and Kpn I restriction sites of the pEGFP-C1 vector, the EcoRI and EcoR V restriction sites of the pCAGGS-Myc / 2HA vector, and the EcoRI and Sal I restriction sites of the p3xFlag-CMV-7.1 vector, respectively. All PCR products were ligated into the vector plasmids via homologous recombination using 2×MultiF Seamless Assembly Mix. For mutant expression plasmids, mutant PCR primers for multi-species IFNAR2 were designed based on the QuickChange primer design method (https: / / www.agilent.com) (see Table S1). Using pEGFP-C1-pIFNAR2 as a template, mutant PCR was performed using KOD plus neo polymerase. After digestion with Dpn I, the PCR products were transformed into competent DMT E. coli, and the resulting mutants were sequenced for verification.

[0033] Cell lines: Porcine alveolar macrophage cell line (3D4 / 21), porcine kidney cell line (PK-15), primary porcine alveolar macrophages (PAM), human embryonic kidney 293T cells (HEK293T), and rhesus monkey kidney cells (MA104) were all preserved by the Animal Infection and Immunology Laboratory of Yangzhou University. Among them, 3D4 / 21 cells (ATCC cat # CRL-2843), PK-15 cells (ATCC cat # CCL-33), HEK293T cells (ATCC cat # CRL-3216), and MA104 cells (ATCC cat # CRL-2378.1) were purchased from ATCC, and primary alveolar macrophages (PAM) were isolated from the bronchoalveolar lavage fluid of experimental pigs.

[0034] Strains: Escherichia coli (E. coli) DH5α competent bacteria were stored in the Animal Infection and Immunology Laboratory of Yangzhou University.

[0035] (2) Reagents and viruses

[0036] Cell culture medium DMEM and RPMI were purchased from Hyclone, Inc., USA. Fetal bovine serum was purchased from Beijing Zhong Sheng Aobang Biotechnology Co., Ltd. Penicillin-streptomycin bispecific antibody (100×, P1400) was purchased from Beijing Solarbio Science & Technology Co., Ltd. TRIPURE Reagent was purchased from Beijing Adley Biotechnology Co., Ltd. Lipo2000 transfection reagent was purchased from Thermo Fisher Scientific, Shanghai. High-fidelity enzyme TSINGKE TSE101 Gold Mix (green) (TSE102) and pClone007 Versatile Simple Vector Kit (TSV-007VS) were purchased from Beijing Qingke Biotechnology Co., Ltd. The Dual Luciferase Reporter Assay Kit (DL101-01), the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper), R312-01, the gel extraction / DNA purification kit (DC301-01), the plasmid extraction kit (DC201-01), the universal high-sensitivity dye-based quantitative PCR kit (Q711), and the 180 kDa Prestained Protein Marker (MP102-01) were purchased from Nanjing Novizan Biotechnology Co., Ltd. The KOD-Plus-Neo high-fidelity DNA polymerase (KOD-401) was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd. Restriction endonucleases Dpn I (R0176V), Bgl II (R0144V), EcoR I (R0101V), Kpn I (R3142V), Bbs I (R3539S), EcoR V (R3195V), and Sal I (R0138V) were purchased from NEB (Beijing) Co., Ltd. Homologous recombinase 2X MultiF Seamless Assembly Mix (RK21020) was purchased from Wuhan Aibote Biotechnology Co., Ltd. Poly I:C-LMW, 2'3'-cGAMP, and poly dA:dT were purchased from InvivoGen. Human IFN-α2b (C600036-0020) and Human IFN-β1b (C600038-0002) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Human IL-28B (CS26) and Human IL-29 (C668) were purchased from Nearshore Protein Technology Co., Ltd. Protein A / GPLUS agarose beads were purchased from Santa Cruz Biotechnology. T4 DNA ligase was purchased from Thermo Fisher Scientific, Inc.4',6-Diamidinyl-2-phenylindole (DAPI) staining solution (C1005) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. All other chemical reagents were of analytical grade and purchased commercially.

[0037] Rabbit monoclonal antibodies: HA (3724S), TBK1 (3504S), p-TBK1 (5483S, Ser172), IRF3 (11904S), FLAG (14793), GFP (2956), and β-actin (5057) were purchased from Cell Signaling Technology, USA. Phosphorylated 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. Phosphorylated STAT1-Y701 rabbit monoclonal antibody (AP0054) and IFNAR2 rabbit polyclonal antibody (A1769) were purchased from Wuhan Aibote Biotechnology Co., Ltd. STAT1 rabbit monoclonal antibody (P42224) was purchased from ZhengNeng Biotechnology. mCherry rabbit polyclonal antibody (ab183628) and Alexa Fluor® 568-labeled goat anti-rabbit IgG H&L (ab175471) were purchased from Abcam, UK. Rabbit polyclonal antibody Myc (16286-1-AP) and mouse monoclonal antibody GAPDH (60004-1-Ig) were both purchased from Wuhan Sanying Biotechnology Co., Ltd. Mouse monoclonal antibodies HA (HT301-01), GFP (HT801-01), and FLAG (HT201-01) were all purchased from TransGen Biotech Ltd. HRP-labeled highly cross-linked goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L) secondary antibodies were purchased from Sangon Biotech (Shanghai) Co., Ltd. Mouse anti-pS273R, p30, and p72 monoclonal antibodies were prepared and stored by the Animal Infection and Immunology Laboratory of Yangzhou University.

[0038] All ASFV infection experiments were conducted in the Animal Biosafety Level 3 (ABSL-3) laboratory at Yangzhou University, 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 and ASFV-mCherry) strains were used. Specifically, the ASFVΔMGF100-1R strain was obtained by deleting the MGF100-1R gene from the isolated ASFV YZ-1 strain (https: / / doi.org / 10.1186 / s44149-024-00130-1), resulting in ASFV-GFP and ASFV-mCherry strains. All strains were stored in the Animal Biosafety Level 3 (ABSL-3) laboratory at Yangzhou University.

[0039] The materials and reagents mentioned in this invention are available to the public through commercial channels both domestically and internationally, and will not be described in detail here.

[0040] Table S1. Primer sequences for amplification and PCR amplification of the mutant gene.

[0041]

[0042] The technical solution of the present invention will be further described in detail below with reference to the embodiments: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0043] Example 1

[0044] (1) pS273R simultaneously inhibits the activation of the JAK-STAT signaling axis in both DNA and RNA receptor pathways.

[0045] African swine fever infection has been shown to activate DNA and RNA signaling pathways. Therefore, we investigated the effects of pS273R, a key protease, on the activation of both DNA and RNA pathways. Through lentiviral packaging, infection, and selection, a stable 3D4 / 21 cell line expressing pS273R (i.e., an immortalized porcine alveolar macrophage cell line) was constructed. 3D4 / 21 cells were stimulated with various agonists, including poly dA:dT (2 μg / ml), poly I:C (2 μg / ml), HSV-1 (MOI=0.1), and VSV (MOI=0.01), by transfection or viral infection. The results of the luciferase reporter assay showed that the presence of pS273R significantly inhibited the activity of IFN-β and ISRE promoters activated by DNA or RNA pathway activation (see [link to luciferase reporter assay]). Figure 1(A). Transcripts of multiple downstream antiviral genes were measured, and qRT-PCR results showed that pS273R expression reduced the expression of IFN-β, ISG, and inflammatory genes, especially various ISGs (see A). Figure 1 (B). Analysis using immunoblotting revealed that pS273R not only inhibited IRF3 phosphorylation mediated by DNA and RNA pathway activation, but also significantly reduced STAT1 phosphorylation (see [link to study]). Figure 1 (C).

[0046] (2) pS273R directly inhibits IFN-I / III-mediated ISG expression.

[0047] To determine whether the inhibition of STAT1 phosphorylation by ASFV pS273R is due to its inhibition of IFN-β production, we used 3D4 / 21 cells with IRF3 knockout, transfected with mCherry-C1-pS273R (1 μg). Luciferase reporter assays using IFN-I / III stimulation showed that overexpression of pS273R directly inhibited the activity of the IFN-I / III-activated ISRE promoter (see [link to study]). Figure 2 The qRT-PCR results also showed that pS273R significantly reduced the transcription of multiple ISGs stimulated by IFN-I / III (see A). Figure 2 (B). In IRF3 - / - In 3D4 / 21 cells transfected with different concentrations of the eukaryotic expression plasmid mCherry-C1-pS273R (0, 0.25, 0.5, 1 μg), Western blotting results showed that pS273R inhibited IFN-I-activated STAT1 phosphorylation in a dose-dependent manner (see...). Figure 2 (C and D). Furthermore, overexpression of pS273R in the porcine kidney cell line PK-15 yielded IRF3. - / - Consistent results in 3D4 / 21 cells (see) Figure 2 (E). Limited by the transfection efficiency of the two cell types, when pS273R is stably expressed in cells, the inhibition of the IFN-I / III activated JAK-STAT pathway is more significant (see E). Figure 2 (F).

[0048] (3) pS273R interacts with IFNAR2, cleaving IFNAR2 and inhibiting JAK-STAT signal transduction.

[0049] To investigate the molecular mechanism by which pS273R inhibits the IFN-I / III-activated JAK-STAT signaling axis, four IFN receptors, two kinases, and three transcription factors associated with it were cloned. These nine proteins (0.5 μg) were co-transfected with the pS273R (0.5 μg) expression plasmid into 293T cells, and their interactions with pS273R were examined. Immunoprecipitation results showed that pS273R significantly interacted with multiple proteins, including IFNAR2, IFNLR1, IRF9, and STAT2, and also had a weak interaction with STAT1 (see [link to immunoprecipitation study]). Figure 3 (A and B). Subcellular localization of pS273R with these proteins was observed using laser confocal microscopy. Consistent with the results of immunoprecipitation, pS273R showed significant colocalization with IFNAR2, IFNLR1, IRF9, STAT1, and STAT2 (see A and B). Figure 3 (C and D). Because pS273R belongs to the SUMO-like protease family, its cleavage effect on nine proteins in the JAK-STAT signaling axis was further investigated. Westen blotting results showed that pS273R cleaves IFNAR2, producing an N-terminal fragment of approximately 70 kDa fused with GFP (see C and D). Figure 3 (E). Except for IFNAR2, pS273R had no cleavage effect on the other 8 proteins, nor did it reduce the expression of these proteins (see E). Figure 3 (E and F).

[0050] Further, by overexpressing different concentrations of pS273R (0, 0.25, 0.5, 1 μg) in 293T cells, or in IRF3... - / - 3D4 / 21 cells were infected with different doses of ASFV-GFP (MOI=0, 0.01, 0.1, 1) to examine the cleavage activity of pS273R. Westen blotting results showed that the ASFV pS273R protease cleaved exogenous and endogenous IFNAR2 in a concentration-dependent manner (see...). Figure 4 (A and B). In the ASFV infection assay, it was determined that pS273R cleavage of IFNAR2 produced a fragment with an actual size of 40-55 kD, which is consistent with the cleavage results of IFNAR2 overexpressed with GFP (see A and B). Figure 4(B). IFNAR2 plays a crucial role in IFN-I-mediated JAK-STAT signaling activation. In fact, IFN-I first binds to IFNAR2 with high affinity, thereby exposing its binding epitope to IFNAR1, forming a tighter IFNAR2-IFN-IFNAR1 complex. However, immunoprecipitation results showed that the interaction between IFNAR2 and IFNAR1 was significantly reduced in the presence of pS273R, and this inhibitory effect was independent of IFN stimulation and binding (see B). Figure 4 Furthermore, pS273R cleavage of IFNAR2 also significantly inhibited its interaction with downstream kinase JAK1 (see C). Figure 4 (D).

[0051] (4) pS273R cleaves IFNAR2 in an enzyme activity-dependent manner.

[0052] pS273R is mainly composed of two domains, AD and CD. The catalytic triplet C232, H168, N187 at the C-terminal active site is a key site for its SUMO-like protease activity. To further verify whether pS273R's cleavage of IFNAR2 depends on its enzyme activity, two separate fragments of pS273R (AD and CD) and mutant expression plasmids with three enzyme activity sites were constructed in the mCherry-C1 vector (see...). Figure 5 (A). These truncated or mutant variants were co-transfected with IFNAR2 in 293T cells. Western blotting results showed that IFNAR2 cleavage by pS273R was dependent on the integrity of its structure, and deletion of either AD or CD completely inhibited its IFNAR2 cleavage (see A). Figure 5 (B). Furthermore, mutations at any one of the three active sites constituting the pS273R catalytic center completely inhibited the enzyme activity of pS273R, thus relieving its cleavage of IFNAR2. Co-transfection of the pS273R mutant with IFNAR2 into 3D4 / 21 cells and laser confocal microscopy observation showed that mutations at the enzyme active sites of pS273R did not affect its co-localization with IFNAR2 (see B). Figure 5 (C and D).

[0053] (5) IFNAR2 is an important receptor protein against ASFV infection.

[0054] IFN-I-mediated antiviral effects have been extensively studied in the pathogenesis and treatment strategies of various viral diseases, including influenza IV, porcine reproductive and respiratory syndrome virus (PRRSV), and African swine fever virus. The core of this approach is the induction of expression of hundreds of downstream antiviral interferon-stimulating genes (ISGs), whose products set up barriers at various stages of viral replication (such as viral RNA synthesis and internalization). However, the IFNAR receptor, which serves as a bridge between IFN-I and various ISGs, has received less attention in these antiviral studies, and its role in resisting viral infection, especially ASFV, remains unclear. Therefore, using CRISPR-Cas9 gene editing technology, IFNAR2 in pigs was knocked out and its genotype was sequenced, resulting in the IFNAR2 receptor. - / - and IRF3 - / - IFNAR2 - / - 3D4 / 21 cells (see) Figure 6 (A). Functional identification of the two knockout cell lines was performed, and qRT-PCR results showed that in IFNAR2... - / - In 3D4 / 21 cells, knockout of IFNAR2 did not affect IFN-β and TNF-α transcription following polyI:C and polydA:dT stimulation, or activation of the RNA and DNA pathways, but it significantly reduced ISG15 expression activated by both stimulants (see [link to relevant documentation]). Figure 6 (B). When treated directly with IFN-I and IFN-III, qRT-PCR results showed that the loss of IFNAR2 completely inhibited the transcription of ISG15 activated by IFN-α and IFN-β, but had almost no effect on the activation of ISG15 by the other two IFN-III (see B). Figure 6 (C). In addition, in two other IRF3 plants... - / - IFNAR2 - / - In 3D4 / 21 cells, the same results as with IFNAR2 knockout alone were obtained. Regardless of the presence or absence of IRF3, the loss of IFNAR2 completely inhibited the expression of multiple ISGs stimulated by IFN-I, including ISG15, ISG56, and CXCL10, but did not affect the ISG-stimulating activity of IFN-III (see...). Figure 6 (D).

[0055] To further clarify the role of IFNAR2 in combating ASFV infection, in WT, IFNAR2 - / - IRF3 - / - and IRF3 - / - IFNAR2 - / -Four types of 3D4 / 21 cells were infected with ASFV-GFP (MOI=0.1). The results showed that, without any treatment, ASFV-GFP had low infection efficiency in wild-type 3D4 / 21 cells, while IFNAR2 knockout significantly enhanced the infection efficiency of ASFV-GFP in 3D4 / 21 cells (see [link to study]). Figure 7 Furthermore, when IRF3 is absent, the impaired synthesis of IFN-I in cells significantly reduces the cells' ability to resist viral infection, and the proportion of ASFV-GFP-infected positive cells increases. IFN-α supplementation, in a concentration-dependent manner, restores the cells' ability to resist ASFV-GFP infection, while the absence of IFNAR2 completely inhibits IFN-I-mediated anti-ASFV infection (see AC). Figure 7 (DF). This suggests the crucial role of IFNAR2 in combating viral infections.

[0056] (6) G399 is the key site for IFNAR2 cleavage by pS273R.

[0057] Similar to the processing of SUMO precursors by SUMO proteases, pS273R, as a SUMO-like protease, can specifically cleave target proteins after two consecutive glycine residues (GG). For example, the ASFV precursor proteins pp62 and pp220 have two and four cleavage sites, respectively. Correspondingly, examination of the amino acid sequence of porcine IFNAR2 protein revealed four potential GG motifs that can be cleaved by pS273R (see...). Figure 8 (A). By mutating the second glycine in a continuous glycine sequence to alanine, four mutant expression plasmids for IFNAR2 in the pEGFP-C1 vector, namely G182A, G250A, G340A, and G399A, were obtained (see A). Figure 8 (B). In HEK293T cells, IFNAR2 and four mutants were co-transfected with pS273R. Western blotting results showed that only the G399 mutation completely resisted pS273R cleavage, while the mutations at the other three sites did not affect pS273R cleavage of IFNAR2 (see B). Figure 8 (C). Furthermore, IFA observations showed that individual mutations at these four sites did not affect the interaction and subcellular co-localization of IFNAR2 and pS273R (see C). Figure 8 (D and E). Immunoprecipitation results also showed that the G399 mutation in IFNAR2 not only resisted pS273R cleavage but also restored its ability to bind to JAK1 (see D and E). Figure 8 (F). By co-transfecting IFNAR2 with four mutants and pS273R into IFNAR2. - / -3D4 / 21 cells were stimulated with a combination of IFN-α and IFN-β. qRT-PCR results showed that, compared to wild-type IFNAR2, the complementation of the G399A mutant significantly enhanced the expression of various ISGs mediated by IFN-I and weakened the inhibitory effect of pS273R on IFN-I-induced ISGs (see...). Figure 8 (G).

[0058] The ASFV pS273R protease cleaves IFNAR2 at the G399 site, mimicking the two fragments of cleaved porcine IFNAR2. These fragments, combined with the two pS273R fragments, were then used to explore the key regions of their interaction. In HEK293T cells, either the truncated pS273R fragment was co-transfected with IFNAR2, or vice versa. Immunoprecipitation results showed that the interaction between pS273R and IFNAR2 depends on the integrity of their structure; the deletion of either fragment completely eliminates the ability to bind to IFNAR2 (see [link to relevant documentation]). Figure 9 (A). The cleaved N-terminus of IFNAR2 is the core region for its interaction with pS273R. The simulated IFNAR2-N not only has the exact same size as the cleaved N-terminus of IFNAR2, but it can also interact significantly with pS273R on its own (see A). Figure 9 (B). Further transfection and refilling of the two IFNAR2 fragments into IFNAR2 was performed. - / - 3D4 / 21 cells were examined for ISG activation activity. The results showed that the cleaved N-terminal fragment of IFNAR2 significantly reduced ISG induction ability, while the C-terminal fragment completely lost its ISG responsiveness to IFN-I stimulation (see...). Figure 9 (C).

[0059] (7) IFNAR2 loses its ability to resist ASFV infection after being cut.

[0060] To detect the anti-ASFV activity of different IFNAR2 mutants and cleavage fragments, the IRF3 assay was performed. - / - IFNAR2 - / -3D4 / 21 cells were transfected with complement IFNAR2 or its mutants and truncated forms (1 μg). Twelve h post-transfection, cells were stimulated with combined IFN-α and IFN-β, followed by ASFV-GFP infection six h post-stimulation. The effect on ASFV replication was examined. Fluorescence and flow cytometry results showed that IFNAR2 remodeling significantly inhibited ASFV-mCherry infection efficiency. Mutations in G182, G250, and G340 did not alter the antiviral activity of IFNAR2, but the G399 mutation further enhanced the antiviral effect of IFN-I stimulated by IFN-I, significantly reducing the number of ASFV-positive cells. The two cleaved fragments of IFNAR2 barely mediated the anti-ASFV effect of IFN-I, and the ASFV infection efficiency was comparable to that of the empty vector (see [link to original text]). Figure 10 (A and B). Furthermore, Western blotting results showed that ASFV infection significantly cleaved the reconstructed IFNAR2 and the three mutants G182A, G250A, and G340A, while the G399A mutant resisted this cleavage and produced a stronger antiviral effect (see A and B). Figure 10 (C).

[0061] This invention is the first to identify that the porcine IFNAR protein, during ASFV infection, can be specifically cleaved at the G399 site by the ASFV protease pS273R, inhibiting the formation of the IFN-I receptor complex and the production of antiviral ISG, thereby promoting viral replication. Targeting this cleavage site, studies have shown that mutants at this site can completely eliminate the cleavage effect of pS273R, effectively inhibiting African swine fever virus replication and significantly improving resistance to ASFV infection. Furthermore, gene editing technology was used to edit the porcine IFNAR2 gene site to breed African swine fever resistant breeds.

[0062] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A porcine IFNAR2 protein mutant, characterized in that, The mutant is a porcine IFNAR2 protein in which glycine is mutated to alanine at position 399, and the amino acid sequence of the porcine IFNAR2 protein is shown in SEQ ID NO.

1.

2. A polynucleotide molecule encoding the porcine IFNAR2 protein mutant of claim 1.

3. A carrier comprising the polynucleotide molecule of claim 2.

4. A host cell, characterized in that, It includes the vector of claim 3 or the chromosome integration of the polynucleotide molecule of claim 2.

5. The use of the porcine IFNAR2 protein mutant of claim 1, the polynucleotide molecule of claim 2, the vector of claim 3, or the host cell of claim 4 in the fight against African swine fever virus infection.

6. The use of the porcine IFNAR2 protein mutant of claim 1, the polynucleotide molecule of claim 2, the vector of claim 3, or the host cell of claim 4 in the preparation of a medicament for the prevention and / or treatment of African swine fever virus infection.

7. The use of the porcine IFNAR2 protein mutant of claim 1, the polynucleotide molecule of claim 2, the vector of claim 3, or the host cell of claim 4 in the breeding of antiviral pigs.

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