Use of rad18 in type i interferon regulation

Abstract

This invention discloses the application of RAD18 in the regulation of type I interferon, belonging to the biomedical field. This invention reveals the complete molecular mechanism by which RAD18, as a key negative regulator of the IRF3-IFN signaling axis, terminates IFNB transcription by phosphorylating IRF3 through K63-type polyubiquitination and mediating its autophagic degradation. Based on this mechanism, this invention proposes a bidirectional regulatory strategy using RAD18 as a molecular switch: inhibiting RAD18 expression or activity can enhance type I interferon production for anti-RNA virus infection; promoting RAD18 expression or activity can inhibit type I interferon overexpression for the treatment of systemic lupus erythematosus.

Description

[0001] This application claims priority to the earlier Chinese application, application number 202510953160.6, filed on July 10, 2025; all its contents are part of this invention. Technical Field

[0002] This invention relates to the field of biomedicine, specifically to the application of RAD18 in the regulation of type I interferon. Background Technology

[0003] Type I interferons (IFNs) are core effector molecules in the host's innate antiviral immune response, playing a crucial role in resisting infections from various pathogens, including RNA and DNA viruses. During viral infection, pattern recognition receptors (such as RIG-I, MDA5, TLR3 / 7 / 9) recognize viral nucleic acids and activate the key transcription factor, interferon regulatory factor 3 (IRF3), through a downstream signaling cascade. IRF3 is phosphorylated by kinases (such as TBK1 and IKKε) to form a homodimer, which translocates into the cell nucleus and binds to the interferon-β (IFN-β) and interferon-stimulated response elements (ISREs) on the promoters of interferon-stimulated genes (ISGs), initiating the transcription and expression of type I interferons, thereby establishing an antiviral immune state.

[0004] However, the production of type I interferon must be strictly regulated. Excessive interferon response can lead to tissue damage, cytokine storms, and even autoimmune diseases; while insufficient interferon response can cause persistent viral replication and exacerbate infection. The balance between IRF3 activation and degradation is crucial for maintaining immune homeostasis. Existing research indicates that IRF3 degradation involves the ubiquitin-proteasome system and the autophagy-lysosome pathway, but the selective degradation mechanisms of IRF3 in different functional states and its key regulatory molecules are not fully understood.

[0005] In the field of antiviral therapy, enhancing the host's innate immune response is one of the important treatment strategies. For RNA virus infections (such as influenza virus, SARS-CoV-2, Zika virus, etc.), there is currently a lack of broad-spectrum and effective targeted drugs, and treatment largely relies on symptomatic supportive therapy or vaccine prevention. At present, strategies to enhance antiviral capabilities by regulating the host's immune response are still in the exploratory stage, and there is an urgent need to identify new molecular targets and mechanisms of action.

[0006] In the field of autoimmune diseases, the overexpression of type I interferon is closely related to the occurrence and development of various diseases. Systemic lupus erythematosus (SLE) is a typical type I interferon-driven autoimmune disease, and patients often exhibit an "interferon signature," which is an abnormally high expression of multiple interferon-stimulated genes. SLE patients show elevated IRF3 phosphorylation levels in peripheral blood mononuclear cells. IFNB1 / IFNA Increased mRNA expression suggests that the IRF3-IFN signaling axis is involved in the pathogenesis of SLE. Currently, the treatment of SLE mainly relies on glucocorticoids, antimalarial drugs, and immunosuppressants. Although these drugs can control the disease to some extent, they have problems such as significant side effects and poor efficacy in some patients. There is an urgent need to develop new targeted therapy strategies against the interferon pathway.

[0007] Therefore, identifying regulatory factors that can regulate phosphorylated IRF3 stability and IFNB transcription, and elucidating their molecular mechanisms, is of great significance for understanding the balance between antiviral immunity and autoimmunity, as well as for developing treatment strategies for related diseases. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides the application of RAD18 in the regulation of type I interferon, belonging to the biomedical field. This invention reveals the complete molecular mechanism by which RAD18, as a key negative regulator of the IRF3-IFN signaling axis, terminates IFNB transcription by phosphorylating IRF3 through K63-type polyubiquitination and mediating its autophagic degradation. Based on this mechanism, this invention proposes a bidirectional regulatory strategy using RAD18 as a molecular switch: inhibiting RAD18 expression or activity can enhance type I interferon production for anti-RNA virus infection; promoting RAD18 expression or activity can inhibit type I interferon overexpression for the treatment of systemic lupus erythematosus.

[0009] On one hand, the present invention provides the use of reagents that regulate RAD18 expression or activity in the preparation of drugs for bidirectional regulation of type I interferon expression, the use including: enhancing type I interferon expression using reagents that inhibit RAD18 expression or activity; or inhibiting type I interferon expression using reagents that promote RAD18 expression or activity.

[0010] Furthermore, the bidirectional regulation is achieved through a RAD18-mediated p-IRF3 ubiquitination degradation mechanism.

[0011] Furthermore, the RAD18 phosphorylates IRF3 by K63-type polyubiquitination modification.

[0012] Furthermore, the ubiquitinated p-IRF3 is recognized by the autophagy receptor OPTN and recruited to autophagosomes for degradation, thereby negatively regulating the transcriptional expression of type I interferon.

[0013] This invention further reveals the crucial role of the autophagy receptor OPTN in RAD18-mediated p-IRF3 degradation, forming the RAD18-OPTN-p-IRF3 cascade regulatory axis, and provides the application of the E3 ubiquitination ligase RAD18 in regulating interferon expression. By regulating the expression or activity of RAD18, interferon expression levels can be altered. Specifically, RAD18 ligates with... IFNB1 The phosphorylated IRF3 dimer binds to the promoter and initiates K63-type polyubiquitination at lysine 193 of IRF3, promoting the phosphorylation of IRF3 from... IFNB1 The promoter dissociates and degrades via autophagy, thereby terminating IFNB transcription. Therefore, inhibiting RAD18 expression or activity enhances type I interferon production, while promoting RAD18 expression or activity inhibits type I interferon production. The interferon is preferably IFN-β and / or IFN-α.

[0014] Furthermore, the inhibition is achieved by one of the following methods: using an inhibitory nucleic acid molecule, knocking out or knocking down the RAD18 gene, using a CRISPR-Cas9 gene editing system, or using a small molecule compound that blocks the binding of RAD18 to p-IRF3.

[0015] The common feature of the above-mentioned inhibition strategies is the blocking of RAD18's recognition, ubiquitination, or degradation functions of phosphorylated IRF3. Specifically, repressive nucleic acid molecules (such as siRNA, shRNA, and antisense oligonucleotides) can downregulate RAD18 protein expression levels through RNA interference or translational arrest mechanisms; gene editing systems (such as CRISPR-Cas9) can achieve functional knockout or knockdown of the RAD18 gene; small molecule compounds can block RAD18's ubiquitination of p-IRF3 by competitively binding to the SAP domain of RAD18 or interfering with its protein-protein interaction with p-IRF3. Any of these methods can block the RAD18-mediated p-IRF3 degradation pathway, thereby enhancing the transcriptional expression of type I interferons.

[0016] Furthermore, the promotion is achieved by one of the following methods: using a promoting nucleic acid molecule, using a RAD18 overexpression vector, or using a small molecule compound that promotes the binding of RAD18 to p-IRF3.

[0017] The common feature of the aforementioned promotion strategies lies in enhancing RAD18's recognition, ubiquitination, and degradation functions of phosphorylated IRF3. Specifically, promoting nucleic acid molecules (such as mRNA and small activating RNA) can upregulate RAD18 protein expression levels; RAD18 overexpression vectors achieve stable high expression of RAD18 in cells through exogenous gene introduction; and small molecule compounds promote RAD18's ubiquitination of p-IRF3 by allosterically regulating or enhancing the affinity of RAD18's SAP domain for p-IRF3. Any of these methods can enhance the RAD18-mediated p-IRF3 degradation pathway, thereby inhibiting the over-transcriptional expression of type I interferon.

[0018] On the other hand, the present invention provides the use of reagents that inhibit RAD18 expression or activity in the preparation of drugs that enhance type I interferon expression to combat RNA virus infection.

[0019] Based on the mechanism that inhibiting RAD18 can enhance the production of type I interferon, this type of reagent maintains the transcriptional activity of IFNB by blocking the ubiquitination degradation of phosphorylated IRF3 by RAD18, thereby enhancing the body's antiviral immunity.

[0020] Furthermore, the RNA virus is selected from any one or more of the following: SARS-CoV-2, vesicular stomatitis virus, H1N1 influenza virus, or Zika virus.

[0021] In another aspect, the present invention provides the use of reagents that promote RAD18 expression or activity in the preparation of drugs that reduce the overexpression of type I interferon in patients with systemic lupus erythematosus.

[0022] Based on the mechanism that promoting RAD18 can inhibit the production of type I interferon, this class of reagents accelerates the termination of IFNB transcription by enhancing the ubiquitination degradation of phosphorylated IRF3 by RAD18, thereby inhibiting the overactivated interferon response.

[0023] Furthermore, the systemic lupus erythematosus patient is a patient with active systemic lupus erythematosus, whose RAD18 protein level in monocytes is lower than that in healthy controls, and IFNB1 / IFNA mRNA levels were higher than in healthy controls.

[0024] The present invention has the following beneficial effects: 1. The E3 ubiquitination ligase RAD18 was identified as a key negative regulator of IRF3 stability and IFNB transcription. The complete molecular mechanism by which RAD18 phosphorylates IRF3 through K63-type ubiquitination and mediates its autophagic degradation was elucidated, providing a new perspective for understanding the balanced regulation of antiviral immunity and autoimmune diseases.

[0025] 2. Based on the regulatory mechanism of the RAD18-p-IRF3-IFN axis, this invention proposes for the first time a bidirectional application strategy of RAD18 as a molecular switch: on the one hand, inhibiting RAD18 expression or activity can enhance the production of type I interferon for anti-RNA virus infection; on the other hand, promoting RAD18 expression or activity can inhibit the overexpression of type I interferon for the treatment of systemic lupus erythematosus. This invention achieves bidirectional application of the same target.

[0026] 3. The antiviral effect of inhibiting RAD18 in various RNA virus infections was verified. Rad18 f / f In Lysm-Cre mice, RAD18 deficiency significantly improved resistance to VSV infection, increased serum IFN-β levels, reduced tissue viral load, and alleviated lung damage. In human macrophages, knockdown of RAD18 expression significantly inhibited the replication of H1N1, Zika virus, and the SARS-CoV-2 Omeprone variant. IFNB1 / IFNA / ISGs The expression level was significantly increased. These results indicate that RAD18 inhibitors have the potential for broad-spectrum anti-RNA virus clinical application.

[0027] 4. The clinical relevance of RAD18 in systemic lupus erythematosus (SLE) was validated. The level of RAD18 protein in monocytes of patients with active SLE was significantly lower than that in healthy controls, while phosphorylated IRF3 and... IFNB1 / IFNA The mRNA level was significantly elevated, and the RAD18 level was negatively correlated with both. These results indicate that the RAD18 protein level is negatively correlated with the level of type I interferon in patients with systemic lupus erythematosus (SLE), and promoting RAD18 expression or activity may be a potential therapeutic strategy for SLE. Attached Figure Description

[0028] Figure 1 For example, in Example 1, the IRF3 degradation pathway was identified; Figure 2 This serves as supplementary verification of the IRF3 degradation pathway in Example 1; Figure 3 Example 2: Screening of RAD18 and its interaction with p-IRF3; Figure 4 This serves as supplementary validation of RAD18 as a potential E3 ligase for p-IRF3 in Example 2. Figure 5 Example 2: RAD18 binds to p-IRF3 and catalyzes K63-type polyubiquitination; Figure 6 Example 2: Relationship between IRF3 phosphorylation and ubiquitination; Figure 7Example 3: RAD18 as a negative regulator of the type I interferon response center; Figure 8 Example 3 compares the functions of RAD18 with other E3 ligases and Pin1; Figure 9 For Example 4 RAD18 in IFNB1 p-IRF3 binds to the promoter; Figure 10 Example 4: Interaction between IRF3 and RAD18 and degradation of RAD18; Figure 11 Example 4 RAD18 termination IFNB1 Transcription; Figure 12 Example 4: RAD18 regulates the transcriptional activity and ubiquitination of IRF3; Figure 13 Example 4: RAD18 regulates IRF3-p300 interaction and IRF3 / IRF7 / IRF5 transcriptional activity; Figure 14 This serves as verification of the mechanism of OPTN-mediated ubiquitination and autophagy degradation of p-IRF3 in Example 5; Figure 15 This serves as supplementary verification of OPTN-mediated autophagic degradation of ubiquitinated p-IRF3 in Example 5; Figure 16 Example 6: Application of RAD18 in combating RNA virus infection; Figure 17 Example 6 illustrates the antiviral and anti-EAE effects of RAD18 deficiency and Irf3-K188R mutation; Figure 18 Example 7: RAD18 expression and clinical relevance in systemic lupus erythematosus. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are only for explaining the present invention and are not intended to limit the scope of protection of the present invention. Experimental methods in this invention that do not specify specific conditions are all conventional methods; reagents that do not specify a specific source are all commercially available conventional reagents.

[0030] Example 1: Identification of IRF3 degradation pathway To determine the degradation pathway of IRF3, an RNA virus infection model was used in the study. Mouse peritoneal macrophages were infected with vesicular stomatitis virus (VSV), and HEK293T cells were infected with Sendai virus (SeV, provided by Professor Xu Pinglong of Zhejiang University).

[0031] In uninfected mouse peritoneal macrophages (PMs), the IRF3 protein underwent some degradation upon administration of cyclohexylimide (CHX) and was inhibited by proteasome inhibitors but not by autophagy inhibitors. Figure 1 a, Figure 2 a). High doses of VSV infection lead to a sharp decrease in p-IRF3 protein levels, a phenomenon that can be prevented by autophagy inhibitors but not by proteasome inhibitors. Figure 1 b, c). The two states of human IRF3 (unphosphorylated and phosphorylated) exhibit similar degradation rates. Figure 2 IRF3-5A protein accumulated after treatment with a proteasome inhibitor, while IRF3-5D protein accumulated after treatment with an autophagy inhibitor. Figure 2 Therefore, unphosphorylated IRF3 is primarily degraded in the proteasome, while phosphorylated IRF3 is degraded via the autophagy-lysosome pathway.

[0032] Furthermore, upon infection with weak stimuli (including low doses of RNA viruses, LPS, or HSV-1), phosphorylated IRF3 can be dephosphorylated by PP2A. The decrease in p-IRF3 is almost entirely offset by Calyculin A (a PP2A inhibitor), but not by autophagy inhibitors. Figure 2 Conversely, Calyculin A had no effect on the decrease in p-IRF3 induced by high-dose VSV infection (ik). Figure 1 b).

[0033] The degradation of p-IRF3 in autophagosomes has been investigated by conditional knockout of autophagy-related genes. Atg7 f / f Lysm- Cre or Atg3 f / f Lysm-Cre This was confirmed in peritoneal macrophages (PMs) and bone marrow macrophages (BMDMs) of mice. The development of these autophagy-deficient macrophages was not affected. Following high-dose VSV infection, p-IRF3 accumulation was significantly higher in autophagy-deficient macrophages than in the control group. Figure 1 d, Figure 2 ln). Eight hours post-infection, accumulated IRF3 protein was in a dimer form ( Figure 1 e), while 16 h post-infection, they are mainly distributed in the cytoplasm. Immunofluorescence co-localization assays showed that p-IRF3 and the autophagy marker LC3B co-localized in the cytoplasm (e), while 16 h post-infection, they were mainly distributed in the cytoplasm. Figure 2 o, p, Figure 1 f).

[0034] Cycloheximide inhibition assays confirmed that the degradation of p-IRF3 was blocked in macrophages lacking autophagy. Figure 1 g, h, Figure 2 q, r). Real-time PCR detection of IRF3 mRNA levels excluded the influence of autophagy on gene expression ( ). Figure 1 i).

[0035] The above results indicate that unphosphorylated IRF3 is mainly degraded via the proteasome pathway, while phosphorylated and activated IRF3 is degraded via the autophagy-lysosome pathway.

[0036] Example 2: Screening of RAD18 and verification of its interaction with p-IRF3 Phosphorylated IRF3 needs to be degraded promptly after initiating IFNB transcription to terminate excessive interferon production, but the key E3 ubiquitination ligase mediating this process remains unclear. To identify the key E3 ubiquitination ligase regulating p-IRF3 degradation, this study used liquid chromatography-tandem mass spectrometry to analyze proteins interacting with IRF3 before and after viral infection.

[0037] 1. Mass spectrometry identification of RAD18 The conclusion that p-IRF3 degradation depends on ubiquitination modification is supported by the following evidence: ubiquitination levels increase after autophagy is blocked. Figure 3 a, Figure 4 a), and IRF3, Ub, and LC3B exhibit co-localization ( Figure 3 b). To investigate the key E3 ubiquitin ligase responsible for p-IRF3 protein ubiquitination, flag-tagged IRF3 complexes were purified from infected or uninfected HEK293T cells and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS / MS) and label-free quantification techniques.

[0038] HEK293T cells were transfected with the Flag-IRF3 plasmid (human IRF3 cDNA as shown in SEQ ID NO.1), and cells were collected 48 h later. To fully activate Flag-IRF3, HEK293T cells were co-transfected with the IKKε plasmid, and after 24 h, cells were infected with SeV and cultured for another 24 h. Then, bafimycin A1 (BafA1, 200 nM, GC17597, GLPBIO) was added 12 h before cell collection. Three biological replicates were set up for each group. Cell lysates were immunoprecipitated with anti-Flag antibody, and proteins interacting with IRF3 were identified by liquid chromatography-tandem mass spectrometry. Protein gel blocks were reduced, alkylated, and digested with trypsin overnight. The resulting tripeptides were separated by a reverse-phase C18 column (Agela Technology, China) and directly connected to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA). Peptide identification and quantification were performed using MaxQuant software (v1.6.0.1), and peptide abundance data were processed using R software (version 4.2.2) and MSstats software package (version 3.18.1).

[0039] Within 24 hours post-infection, all Flag-IRF3 proteins were phosphorylated and formed dimers. Figure 4 b). RAD18 is the most abundant E3 ubiquitin ligase in the SeV virus-infected cell complex, and its specific peptides are also the most abundant. Figure 3 c, d). Among the candidate E3 ubiquitin ligases, RAD18 had the highest absolute content. Other E3 ubiquitin ligases detected included PRPF19, TRIP12, RNF2, and TRIM25. Figure 4 c).

[0040] 2. Functional verification of RAD18 in regulating p-IRF3 degradation Overexpression of RAD18 promoted the ubiquitination and degradation of p-IRF3 and inhibited the activation of IFN-β and the expression of interferon-stimulated response element reporter genes, while other candidate E3 ubiquitin ligases did not have this effect. Figure 4 dg). RAD18 deficiency prevents the degradation of p-IRF3, while the wild-type (WT) RAD18 (as shown in SEQ ID NO.2) is reintegrated into Rad18 - / - In bone marrow macrophages, the degradation of p-IRF3 was restored ( Figure 4 h, Figure 3 e). Therefore, RAD18 is an E3 ubiquitin ligase capable of degrading p-IRF3.

[0041] To verify the necessity of RAD18's E3 ubiquitin ligase activity in regulating p-IRF3 degradation, this invention further constructed ubiquitination-deficient mutants RAD18-C28F (with a key cysteine ​​residue at position 28 of the RING domain mutated to phenylalanine, as shown in SEQ ID NO.3) and RAD18ΔRING (with a RING domain deletion mutation, as shown in SEQ ID NO.4). Wild-type RAD18, RAD18-C28F, or RAD18ΔRING were then reintroduced via a lentiviral system. Rad18 - / - In BMDMs, the degradation of p-IRF3 was examined after infection with VSV (MOI=10). Immunoblotting results showed that wild-type RAD18 could completely restore p-IRF3 degradation, while neither RAD18-C28F nor RAD18ΔRING mutants could restore p-IRF3 degradation. Figure 3 e). Ubiquitination experiments in HEK293T cells further confirmed that the RAD18-C28F mutant lost the ability to catalyze IRF3 ubiquitination (e). Figure 3 j). The above results indicate that the ubiquitin ligase activity of RAD18 is essential for its mediating p-IRF3 degradation.

[0042] Blocking autophagy can stop the RAD18-mediated degradation of p-IRF3 protein. Figure 4 i, j). In cells with the RAD18 gene knocked out, autophagy inhibitors did not further promote the accumulation of p-IRF3 (i, j). Figure 4 k. The levels of p-IRF3 dimer were similar in RAD18 knockout cells and ATG7 knockout cells. Figure 4 Therefore, RAD18 promotes the degradation of p-IRF3 dimer via the autophagy pathway.

[0043] 3. Specific binding of RAD18 to p-IRF3 Experiments confirmed the role of RAD18 in the ubiquitination of p-IRF3 protein. First, reverse immunoprecipitation (co-IP) experiments confirmed that RAD18 specifically binds to p-IRF3. Figure 3 f, Figure 5 a). Immunofluorescence co-localization assays showed that RAD18 and p-IRF3 co-localized in the cell nucleus ( Figure 5 b, Figure 3 g).

[0044] In vitro binding assays confirmed the direct interaction between p-IRF3 and RAD18. Purified Flag-tagged IRF3, RAD18, or their corresponding mutants were mixed with double-stranded ISRE DNA in TBS buffer and incubated at room temperature for 1 h. The mixture was then incubated overnight at 4°C with anti-IRF3 antibody (Proteintech, 66670-1-Ig), followed by incubation with Protein A / G magnetic beads at room temperature for 2 h. After washing three times with 1×TBS buffer, the magnetic beads were boiled in 2×SDS loading buffer and analyzed by Western blotting. The results showed that the direct interaction between p-IRF3 and RAD18 occurred only in the presence of double-stranded ISRE DNA. Figure 5 c, Figure 3 h). Double-stranded DNA oligonucleotides were synthesized by annealing synonymous and antisense single-stranded DNA oligonucleotides to 95°C and then slowly cooling to 25°C in annealing buffer (20 mM Tris-HCl, pH 7.5 and 50 mM NaCl). The single-stranded DNA sequence is SEQ ID NO.5, and the 3×ISRE DNA sequence is SEQ ID NO.6.

[0045] 4. RAD18-catalyzed K63-type polyubiquitination of p-IRF3 To determine the type of ubiquitination modification of p-IRF3 by RAD18, IRF3 was co-expressed with RAD18 and different ubiquitin mutants. HEK293T cells were co-transfected with Flag-IRF3, V5-RAD18, and HA-Ub-K48R or HA-Ub-K63R (or HA-Ub-K63-only) plasmids. 24 h after transfection, cells were infected with SeV to activate IRF3. BafA1 was added before cell collection to block autophagic degradation. Cell lysates were immunoprecipitated with anti-Flag antibody, and the ubiquitination modification type of IRF3 was detected by Western blotting. The results showed that RAD18 mainly catalyzes K63-type polyubiquitination of IRF3 (…). Figure 5 d, e). In vitro ubiquitination assays were used to further verify the E3 ubiquitin ligase activity of RAD18 on p-IRF3. The Ubiquitylation Assay Kit (Abcam, Ab139467) was used according to the manufacturer's instructions. E2 was performed using UBE2A / B, and E3 was performed using purified Flag-RAD18 protein as the substrate, with purified Flag-IRF3 protein as the substrate. The results showed that RAD18 could directly catalyze the K63-type polyubiquitination of IRF3 (…). Figure 3 i).

[0046] Key lysine sites on IRF3 modified by RAD18 were identified through site-directed mutagenesis screening. Thirteen lysine residues common to human and mouse IRF3 were replaced with arginine residues to construct the IRF3-KR mutant. Figure 5 f). HEK293T cells were co-transfected with RAD18 and infected with SeV 24 h after transfection to activate IRF3. BafA1 was added 12 h before cell collection, followed by immunoprecipitation with anti-Flag antibody. IRF3 ubiquitination modification was then detected by Western blotting. Results showed that the IRF3-K77R (as shown in SEQ ID NO.7) and IRF3-K193R mutants (as shown in SEQ ID NO.8) almost completely lost ubiquitination modification. Figure 3 k). Given that the IRF3-K77R mutant cannot enter the cell nucleus ( Figure 5 d) Lys193 was identified as the most important site for RAD18-mediated ubiquitination, which was confirmed by the lack of ubiquitination in mouse IRF3-K188R (mouse IRF3 cDNA as shown in SEQ ID NO. 9, mutant as shown in SEQ ID NO. 10) and the effect of the mutation on the activity of the IFN-β reporter gene. Figure 5 g, h).

[0047] Phosphorylation and nuclear translocation are necessary conditions for ubiquitination of the IRF3 protein. IRF3-5A cannot bind to RAD18 and does not undergo ubiquitination. Figure 6 IRF3-K193R was phosphorylated and bound to RAD18 but failed to be ubiquitinated. Figure 6 df). IRF3-K77L (as shown in SEQ ID NO.11) cannot bind to RAD18 and is not ubiquitinated due to the lack of NLS signal transduction capability. Figure 6 df).

[0048] The above results indicate that RAD18 is an E3 ubiquitin ligase that can specifically bind to phosphorylated IRF3 and catalyze its K63-type polyubiquitination.

[0049] Example 3: Validation of RAD18 as a negative regulator of type I interferon response center To determine the role of RAD18 in RNA virus-induced type I interferon responses, the dynamic changes of p-IRF3 were detected in BMDMs infected with VSV (MOI = 10). The results showed that p-IRF3 peaked 4–5 h post-infection and essentially disappeared 12 h post-infection. Rad18 - / - In BMDMs, p-IRF3 accumulation persists up to 12 hours post-infection. Figure 7ac). Rad18 - / - Mice (strain number: T029438) were provided by Jiangsu Jicui Pharmaceutical Co., Ltd.

[0050] Real-time PCR detection Ifnb1 / Ifna mRNA levels and interferon-stimulated genes (including Isg15 , Rantes , Cxcl10 and Ifit1 The mRNA levels of BMDMs were compared to wild-type BMDMs. Rad18 - / - The expression levels of these genes were significantly increased in BMDMs. Figure 7 d, Figure 8 a). The replication level of VSV was detected by a plaque formation assay, and the results showed... Rad18 - / - Viral replication was significantly suppressed in BMDMs. Figure 7 e).

[0051] RAD18 was compared with other reported E3 ubiquitin ligases (TRIM21, TRIM26, RBCK1, and UBE3C) and the negative regulator Pin1. The binding of each protein to p-IRF3 was detected by co-immunoprecipitation. The results showed that RAD18 and Pin1 selectively bind to p-IRF3, while TRIM21, RBCK1, and UBE3C bind to both p-IRF3 and unphosphorylated IRF3. Figure 8 b, c).

[0052] The ubiquitination levels of IRF3 by various E3 ubiquitin ligases were compared using ubiquitination assays. The results showed that overexpression of RAD18 resulted in the highest level of IRF3 ubiquitination. Figure 7 f). The activities of IFN-β and ISRE reporter genes were detected by a dual-luciferase reporter gene assay. Figure 8 (d, e) The results showed that silencing or loss of function of RAD18 had the most significant inhibitory effect on the degradation of p-IRF3, and led to the strongest activity of IFN-β and ISRE reporter genes. Silencing RAD18 in BMDMs had the following effect: Ifnb1 / Ifna The strongest effects were produced by elevated mRNA levels and decreased VSV replication levels. Figure 7 g, h, Figure 8 f, g).

[0053] These results indicate that RAD18 is the central negative regulator of p-IRF3 protein and type I IFN response.

[0054] Example 4, RAD18 in IFNB1 Validation of the mechanism by which p-IRF3 binds to the promoter and terminates transcription. To investigate the molecular mechanism of RAD18-mediated p-IRF3 degradation, and the relationship between this degradation process and... IFNB1 The relationship with transcriptional regulation was investigated by first assessing the spatiotemporal dynamics of IRF3 in SeV-infected HEK293T cells. Nuclear and cytoplasmic proteins were extracted using a nuclear and cytoplasmic component separation and extraction kit (Beyotime Biotechnology, C500009), and the distribution of IRF3, p-IRF3, and RAD18 in each component was detected by Western blotting. Immunofluorescence results showed that endogenous IRF3 underwent dynamic translocation between the nucleus and cytoplasm at different time points after SeV infection. Figure 10 a). Combining immunoblotting and immunofluorescence results, IRF3 underwent a series of events, including phosphorylation and dimerization in the cytoplasm, nuclear translocation, interaction with RAD18, ubiquitination, extranuclear export, and degradation in autophagosomes. Figure 9 ad, Figure 10 b).

[0055] 1. RAD18 and p-IRF3 in IFNB1 binding on promoter RAD18 and IRF3 were detected using chromatin immunoprecipitation assay. IFNB1 Promoter binding dynamics. ChIP-qPCR experiments were performed using the CUT&RUN kit (Cell Signaling Technology, 86652) according to the manufacturer's instructions. After infection with RNA viruses, cells were washed, bound to magnetic beads, and permeabilized, then incubated overnight at 4°C with appropriate antibodies in wash buffer containing 0.05% digitalis saponins. Cell-magnetic bead complexes were washed twice with digitalis saponin buffer and then mixed with Protein A-MNase for 1 h at 4°C. After cooling on ice, the complexes were incubated with 2 mmol / L calcium chloride to activate Protein A-MNase digestion. DNA digestion was stopped by adding one volume of 2× stop buffer. DNA fragments were extracted using DNA purification buffer and a centrifuge column (Cell Signaling Technology, #14209), and enrichment folds were determined by qRT-PCR as a percentage of total chromatin fraction.

[0056] IRF3 and IFNB1Promoter binding precedes p-IRF3-RAD18 interaction, as demonstrated by the failure of IRF3-R81L (as shown in SEQ ID NO.12) and the IRF3Δ1-57 mutant (as shown in SEQ ID NO.13) (which can enter the nucleus but cannot bind to the promoter) to recruit and ubiquitinate RAD18. Figure 9 e, f, Figure 10 ChIP-PCR showed that IRF3 and RAD18 had similar promoter binding kinetics, peaking at 24 h post-infection and declining sharply at 36 h post-infection. Figure 9 g, h). In IRF3 knockout cells, RAD18 binding to the promoter is almost completely lost ( Figure 9 g). In RAD18 knockout cells, the binding between IRF3 and the promoter was enhanced and remained at a high level until 36 h post-infection. Figure 9 h).

[0057] To determine the interaction domains between IRF3 and RAD18, a series of deletion mutants of RAD18 were constructed. Figure 10 f). HA-IRF3 and various Flag-RAD18 truncated mutants were co-transfected into HEK293T cells, and IKKε was also co-transfected to activate IRF3 phosphorylation. BafA1 (200 nM) was added 12 hours before cell collection, and immunoprecipitation was performed using anti-HA antibody. The binding ability of each RAD18 mutant to IRF3 was detected by Western blotting. The results showed that RAD18ΔSAP completely lost its ability to bind to IRF3, while RAD18ΔRING showed a similar binding strength to wild-type RAD18; notably, RAD18ΔZNF showed a higher binding ability than the wild-type. Figure 9 i, Figure 10 g). This indicates that the SAP domain is a key domain necessary for the binding of RAD18 to p-IRF3, while the ZNF domain may play a suppressive role in this interaction.

[0058] RAD18 protein levels decreased significantly after binding to p-IRF3. Figure 7 a, c, Figure 9 a, d). RAD18 mRNA remains at low levels and increases 44 h post-infection, by which time most RAD18 protein has been degraded. Figure 9 j). Combining wild type and Rad18 - / - Relevant data on BMDMs ( Figure 7 (ad), it can be concluded that: RAD18 degrades p-IRF3 and IFNB1Transcription consistently has a negative effect. Furthermore, proteasome inhibitors can block the degradation of RAD18 (…). Figure 9 k), while MG132 treatment led to a significant accumulation of ubiquitinated RAD18 (k), Figure 10 h). Conversely, the negative regulatory effect of IRF3-driven IFN-β on RAD18 expression was excluded, a finding observed in wild-type and... Ifnar1 - / - This can be demonstrated by similar RAD18 degradation curves in BMDMs. Figure 9 l). IFN-β treatment also did not affect RAD18 protein levels ( Figure 10 i, j).

[0059] 2. RAD18 IFNB1 Termination of transcription The effects of RAD18 on [the target population] were detected by a dual-luciferase reporter gene assay. IFNB1 Effects of transcription. IFN-β and ISRE luciferase reporter plasmids (provided by Professor Li Nan, National Key Laboratory of Medical Immunology), pRL-TK lactose luciferase (Promega, E2241), and designated plasmids were transiently transfected into HEK293T cells using PEI transfection reagent (Polyplus Transfection, 101000053). The amount of DNA at each transfection was normalized by adding an empty pcDNA3.1(-) plasmid (ThermoFisher Scientific) as a control. Luciferase activity in harvested cells was measured using a dual-luciferase assay (Promega, E1910) and analyzed using GloMax. ® The luminescence intensity was measured using a 20 / 20 photometer (Promega, E5311), and the data were analyzed as the ratio of luciferase / lactal luciferase activity.

[0060] The results showed that RAD18 overexpression significantly inhibited viral infection-induced [therapeutic effects]. IFNB1 mRNA synthesis is affected, while the loss of RAD18 leads to... IFNB1 The synthesis increases and is more persistent ( Figure 11 a, b, Figure 12 a, b). Overexpression of RAD18 accelerates IRF3 from IFNB1 Dissociation on the promoter ( Figure 12 c), while RAD18 gene knockout leads to persistent accumulation of IRF3 on the promoter ( Figure 12 d). In RAD18 - / - Reexpression of wild-type RAD18 in cells leads to the dissociation of IRF3 from the promoter and IFNB1The termination of synthesis was reversed, but reexpression of the E3-deficient mutant RAD18-C28F failed to reverse the process. Figure 11 b, Figure 12 b, d).

[0061] Rad18 deletion significantly increases the number of genes containing ISRE sequence promoters (such as...). Rantes, Cxcl10, Ifit1 Irf7 and Il-27p28 At the mRNA level after viral infection ( Figure 11 c, Figure 8 a). The binding of RAD18 to these promoters was confirmed by ChIP experiments, and this binding was significantly inhibited by knocking out IRF3. Figure 11 d). A comparison of the effects of RAD18 and previously reported E3 peptidase and Pin1 on p-IRF3 transcriptional activity showed that knockout of RAD18 prolonged IRF3 transcriptional activity. IFNB1 The residence time on the promoter, while knockout of other E3 peptidases and Pin1 does not have this effect ( Figure 11 e), indicating that RAD18 specifically regulates the residence time of p-IRF3 on the promoter.

[0062] 3. The necessity of RAD18-mediated ubiquitination for IRF3 transcription termination Further investigation was conducted to determine whether RAD18-mediated ubiquitination of p-IRF3 is necessary for the dissociation of IRF3 from the promoter. IRF3-K193R remained persistently bound to the promoter, leading to persistent IFN-β generation. Figure 11 f, Figure 12 g). Whether RAD18 is overexpressed or silenced, the duration of IRF3 (K193R) residence at the promoter and subsequent... IFNB1 Transcription was not affected. Figure 11 f, Figure 12 g). RAD18 gene knockout and K193R mutation eliminated nuclear export of IRF3 ( Figure 11 g, Figure 12 h, i). After infection, wild-type IRF3 and IRF3-5D formed small dots in the nucleus and mainly co-localized with K63-ubiquitin staining, followed by ubiquitinated p-IRF3 leaving the nucleus; IRF3-R81L failed to form small dots in the nucleus; IRF3-K193R formed dots but failed to co-localize with K63-ubiquitin staining; IRF3-NES* (lacking NES signaling) dots co-localized with K63-ubiquitin staining (h, i). Figure 12 i).

[0063] The interaction between IRF3 and the transcriptional coactivator p300 was detected by immunoprecipitation assay. The results showed that the interaction between p-IRF3 and p300 peaked at 24 h post-infection and disappeared after 36 h. Figure 13 a). Overexpression of RAD18 inhibited the interaction between p-IRF3 and p300 / CBP ( Figure 11 h). RAD18 knockout enhances and prolongs this interaction, while reexpression of wild-type RAD18 (but not RAD18-C28F) restores the dissociation of IRF3 from p300 / CBP. Figure 13 b, Figure 11 h). IRF3-K193R is continuously limited by p300 ( Figure 13 c).

[0064] To further confirm the unique role of RAD18 in specifically regulating the retention time of p-IRF3 at the promoter, this invention compared the binding affinity of RAD18 to IRF3 mutants with other reported IRF3 regulators, Pin1 and TRIM21. In HEK293T cells transfected with IKKε, V5-Pin1 (… Figure 12 e) or V5-TRIM21 ( Figure 12 f) Co-immunoprecipitation assays were performed with Flag-IRF3 wild-type or Flag-IRF3-R81L mutant. Results showed that Pin1 and TRIM21 could bind to both IRF3 wild-type and IRF3-R81L mutant. Figure 12 (e, f), while RAD18 binds only to the wild-type IRF3 and not to IRF3-R81L. These results indicate that RAD18 exhibits a unique promoter-dependent binding pattern to IRF3, which differs from the binding patterns of other IRF3 regulators, further suggesting that RAD18 is a key factor specifically regulating the residence time of p-IRF3 on the promoter.

[0065] IRF7 and IRF5, as important transcription factors, can enhance the production of type I IFN in the late stages of viral infection. In VSV-infected BMDMs, immunoprecipitation with anti-RAD18 antibody was used to detect the binding of RAD18 to p-IRF7, p-IRF5, and p-p65. The results showed that RAD18 could specifically bind to p-IRF7 and p-IRF5, but not to another transcription factor, p65. Figure 11 i). Further ubiquitination experiments were conducted to examine the K63-type polyubiquitination modification of IRF7 and IRF5 by RAD18. The results showed that RAD18 significantly promoted the ubiquitination and degradation of IRF7 and IRF5. Figure 11 j, Figure 13d). Chromatin immunoprecipitation assays showed that in Rad18 gene knockout BMDMs, IRF7 and IRF5 were reduced from... IFNB1 Dissociation at the promoter was significantly suppressed. Figure 11 The above results indicate that RAD18 terminates the transcriptional activity of IRF3, IRF7, and IRF5 through a ubiquitination-dependent mechanism.

[0066] Since p-IRF3 induced by weak stimulation can be dephosphorylated by the phosphatase PP2A, this invention further investigated whether RAD18 also participates in the regulation of IRF3 transcriptional activity under weak stimulation conditions. Wild-type and... Rad18 - / - The dynamic changes in p-IRF3 protein levels were detected in BMDMs, or in wild-type and RAD18 knockout HEK293T cells infected with a low dose of SeV (MOI=0.01). The results showed that knockout of the RAD18 gene significantly inhibited the decrease in p-IRF3 after stimulation with a low dose of RNA virus. Figure 13 e, f). Chromatin immunoprecipitation assays further showed that RAD18 knockout prolonged p-IRF3 at [the site of the event]. IFNB1 promoter The time spent on it, and subsequently significantly increased. IFNB1 transcription level ( Figure 13 The above results indicate that RAD18 regulates the transcriptional activity of p-IRF3 before it is degraded or dephosphorylated, further establishing RAD18 as a core negative regulator of the IRF3-IFN axis.

[0067] The above results indicate that RAD18 binds to the site located at IFNB1 p-IRF3 on the promoter mediates its K63-type polyubiquitination, causing p-IRF3 to dissociate from the promoter, thereby terminating the process. IFNB1 Transcription.

[0068] Example 5: Validation of the mechanism of OPTN-mediated ubiquitination and autophagy degradation of p-IRF3 Based on RAD18-mediated p-IRF3 ubiquitination in Example 4, from IFNB1 Following the promoter dissociation and nuclear export, this example further investigates how ubiquitinated p-IRF3 enters the autophagosome for degradation. In SeV-infected HEK293T cells, after 36 h, autophagy cargo receptors NBR1, OPTN, and NDP52 interacted with p-IRF3. Figure 14 a). Two-step co-immunoprecipitation assays showed that OPTN bound to p-IRF3, but not to unphosphorylated IRF3 (a). Figure 14b, c), while NBR1 and NDP52 bind to both unphosphorylated and phosphorylated IRF3 (b, c), Figure 15 a, b). Therefore, OPTN is only combined with IRF3-5D, while NBR1 and NDP52 are combined with IRF3-5A and IRF3-5D ( Figure 14 d, Figure 15 c, d).

[0069] Unlike NBR1 and NDP52, the absence or silencing of OPTN leads to a significant accumulation of p-IRF3 and IRF3 dimers. Figure 15 e, Figure 14 e, f). CHX inhibition experiments also showed that OPTN knockdown inhibited the degradation of p-IRF3 (e, f). Figure 15 f). Therefore, OPTN is the main receptor responsible for p-IRF3 degradation.

[0070] Immunofluorescence colocalization assays showed that OPTN colocalized with p-IRF3 and the autophagy marker LC3B in the cytoplasm. Figure 14 g, Figure 15 g). Blocking autophagy significantly increased the K63-ubiquitination level of p-IRF3 and the interaction between p-IRF3 and OPTN ( Figure 15 h, i). OPTN overexpression accelerates the degradation of p-IRF3, and this degradation is blocked by autophagy inhibitors (h, i). Figure 15 j).

[0071] OPTN primarily recruits substrates by recognizing ubiquitin modifications of the substrate. To clarify the molecular basis of OPTN's specific recognition of p-IRF3, this invention examined the binding affinity of OPTN mutants with different domains to p-IRF3. An OPTNΔUBA mutant (lacking the ubiquitin-related domain) and an OPTN-S473A phosphorylation-deficient mutant were constructed, and their binding affinity to p-IRF3 was detected using an immunoprecipitation assay. The results showed that OPTNΔUBA could not bind to p-IRF3 at all, while the binding affinity of OPTN-S473A to p-IRF3 was significantly reduced. Figure 14 h, Figure 15 k, l). Meanwhile, the IRF3-K77R and IRF3-K193R have almost completely lost their ability to integrate with the OPTN ( Figure 14 i).

[0072] Furthermore, this invention also examined the regulation of p-IRF3-OPTN interaction by RAD18. The results showed that RAD18 overexpression enhanced the interaction between p-IRF3 and OPTN. Figure 14j). RAD18 knockout almost completely suppresses this interaction, while re-introducing wild-type RAD18, but not RAD18-C28F, into the RAD18-C2 ... RAD18 - / - This interaction is restored in the cells. Figure 14 j, k, Figure 15 m). Co-localization of OPTN, GFP-IRF3, p-IRF3, and K63-ubiquitin was observed in the cytoplasm. Figure 15 np). IRF3 mutants, including IRF3-K77L, IRF3-R81L, IRF3-K193R, and IRF3-NES*, cannot bind to OPTN regardless of whether RAD18 is overexpressed. Figure 14 l, Figure 15 q). The absence of OPTN and the blockade of autophagic flux both significantly increased the level of p-IRF3 dimer ( Figure 14 e).

[0073] The above results indicate that the K63-polyubiquitinated p-IRF3 dimer modified by RAD18 in the cell nucleus is exported outside the nucleus, and is specifically recognized by OPTN and recruited to autophagosomes for degradation.

[0074] Example 6: Application of RAD18 in combating RNA virus infection To verify the antiviral function of RAD18 in vivo, macrophage-specific Rad18 knockout mice were constructed. Rad18 f / f Lysm-Cre mice were provided by Professor Cao Xuetao (National Key Laboratory of Medical Immunology). All mice were C57BL / 6 gene-bearing and housed at the Experimental Animal Center of Zhejiang University under specific pathogen-free (SPF) conditions, with ambient temperature controlled at 18-23℃ and humidity controlled at 40-60%, following a 12-hour light / dark cycle. Healthy male or female mice aged 7-10 weeks were used. The animal experimental protocol was approved by the Animal Research Ethics Committee of Zhejiang University (Virus Infection: No. ZJU20210058).

[0075] Control mice ( Rad18 f / f )and Rad18 f / f Lysm-Cre mice were infected with a lethal dose of VSV. Mice survival was recorded daily after infection for 14 consecutive days. Results showed that the survival rate of Rad18 knockout mice was significantly higher than that of control mice. Figure 16a). Serum was collected from mice 12 h post-infection, and IFN-β concentration was measured using an IFN-β ELISA kit (PBL Assay Science, 42400-1) according to the manufacturer's instructions. The results showed that serum IFN-β levels were significantly elevated in Rad18 knockout mice. Figure 16 b). Collect lung, spleen, and liver tissues, detect viral titers using plaque formation assays, and perform real-time PCR detection. Ifnb1 / Ifna and Tnfa The mRNA levels were measured. Results showed reduced viral replication in various tissues of Rad18 knockout mice. Ifnb1 / Ifna Elevated mRNA levels and reduced pulmonary inflammatory infiltration ( Figure 17 af, Figure 16 c).

[0076] To verify the antiviral activity of RAD18 in human cells, CD14-positive monocytes were isolated from peripheral blood of healthy donors and induced to differentiate into macrophages. MojoSort was used. TM Human CD14 positive mononuclear cell isolation kit (Biolegend, 480047) was used according to the manufacturer's instructions and the cells were cultured for 7 days in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Omnimabs, OM625658), 100 U / mL penicillin, 100 mg / mL streptomycin, and 20 ng / mL human M-CSF (Peprotech, 300-25-50UG).

[0077] CD14-positive mononuclear cells were isolated from the peripheral blood of recovered patients with SARS-CoV-2 and induced to differentiate into macrophages. Recovered patients were divided into mild and severe groups based on their SARS-CoV-2 infection status. Patients with SARS-CoV-2 infection were diagnosed based on their clinical symptoms and positive SARS-CoV-2 test results. The classification of "mild infection" and "severe infection" was determined by the attending physician according to the "Diagnosis and Treatment Protocol for Novel Coronavirus Infection (Trial Version 6)" issued by the National Health Commission of China (this study has been approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine, No. 2024-IIT-003). Compared with the mild group, the macrophages of recovered patients in the severe group had higher levels of RAD18 basal protein and mRNA. Figure 16 d, Figure 17 g, h). Following H1N1 infection in vitro, p-IRF3 protein and... IFNB1 / IFNA mRNA levels were lower in the mild case group ( Figure 16 e, f, Figure 17 Correlation analysis showed that RAD18 protein levels were correlated with p-IRF3 and... IFNB1 / IFNA mRNA levels were negatively correlated. Figure 16 g, h, Figure 17 l).

[0078] RAD18 expression was knocked down in macrophages using siRNA, and the cells were infected with H1N1 influenza virus (PR8 / A / 34, provided by Professor Cai Zhijian of Zhejiang University) 48 h later. Results showed that RAD18 knockdown led to p-IRF3 and IFNB1 / IFNA / ISGs Increased mRNA levels Figure 16 ik). Regarding Zika virus infection (provided by Professor Ye Jing of Huazhong Agricultural University), RAD18 - / - A549 cells showed higher levels of p-IRF3 protein and [other proteins] than wild-type A549 cells. IFNB1 / IFNA / ISGs mRNA levels ( Figure 16 A549 cells were infected with Zika virus (infection strength 5). This experiment was supported by Dr. Lin Lin from Shanghai University. For SARS-CoV-2 Omeprón variant infection, RAD18 deletion resulted in decreased p-IRF3 protein levels in A549 cells overexpressing human ACE2 (A549-ACE2). IFNB1 / IFNA / ISGs Increased mRNA levels Figure 16 (oq), A549-ACE2 cells were infected with the SARS-CoV-2 B.1.1.529 (Omecron) variant (infection intensity 0.1, provided by Professor Yao Hangping of Zhejiang University). The experiment was conducted in a biosafety level 3 facility at the First Affiliated Hospital of Zhejiang University.

[0079] In addition, the IRF3-K188R mutation was bred ( Irf3 K188R / K188R The mice were generated by Jiangsu Jicui Pharmaceutical Co., Ltd. using CRISPR-Cas9 technology (C57BL / 6J background). Irf3 K188R / K188R BMDMs in mice showed significant p-IRF3 accumulation. IFNB1 / IFNA / ISGs mRNA levels were significantly increased, while viral replication was reduced compared to the wild type. Figure 16 r, s, Figure 17 m, n). Based on the application of IFN-β in the treatment of multiple sclerosis, Irf3 K188R / K188RExperimental autoimmune encephalomyelitis (EAE) was induced in female mice (6-8 weeks old) by subcutaneous injection of 200 μg of MOG35-55 peptide (Sangon Biotech, T510219), mixed with incomplete Freund's adjuvant (Sigma-Aldrich, F5506), and supplemented with 5 mg / mL of Mycobacterium tuberculosis H-37Ra (BD Biosciences, 231141). On days 0 and 2 post-induction, mice also received two intraperitoneal injections of 500 ng of pertussis toxin (GLPBIO, GC17532). Clinical symptoms of EAE were assessed using a scoring system (0 points: asymptomatic; 1 point: tail loss of tone; 2 points: hind limb weakness; 3 points: hind limb paralysis; 4 points: forelimb paralysis; 5 points: near death). All mice were scored without genotypic information to ensure objectivity. Results showed that, compared to wild-type mice, Irf3 K188R / K188R Mice showed later onset and milder symptoms. Figure 16 t). Histological analysis showed that Irf3 K188R / K188R In mice, inflammatory cell infiltration in the spinal cord was reduced, and myelin loss was lessened. Figure 17 o, p). This animal experimental protocol has been approved by the Animal Research Ethics Committee of Zhejiang University (EAE model: No. ZJU20241009).

[0080] Further comparisons were made between RAD18 and other reported E3 ubiquitin ligases (TRIM21, TRIM26, RBCK1, UBE3C) that regulate IRF3, as antiviral therapeutic targets. Using VSV-infected mouse bone marrow-derived macrophages (BMDMs) as a model, lentiviruses interfering with the expression of different E3 ubiquitin ligases were constructed and introduced into BMDMs. Results showed that reducing RAD18 expression resulted in BMDMs producing the strongest levels of type I IFN and ISGs (including Isg15, Rantes, Cxcl10, and Ifit1), while exhibiting the best inhibitory effect on viral replication. These results indicate that drugs developed using a strategy of reducing RAD18 expression can achieve stronger antiviral therapeutic effects compared to drugs targeting other E3 ubiquitin ligases.

[0081] The above results indicate that inhibiting RAD18 expression or activity can effectively enhance the immune response against RNA viruses.

[0082] Example 7: Application of RAD18 in the treatment of systemic lupus erythematosus To investigate the expression of RAD18 in systemic lupus erythematosus (SLE) and its clinical relevance, peripheral blood was collected from patients with active SLE and healthy controls, and CD14-positive mononuclear cells were isolated.

[0083] For patients with systemic lupus erythematosus (SLE), all patients met the 1997 American College of Rheumatology (ACR) classification criteria for SLE, and lupus disease activity was defined as the SLEDAI-2k score, with patients having active SLE having an SLEDAI > 4. For the treatment-naïve patient group, from October 20 to November 5, 2021, the First Affiliated Hospital of Zhejiang University recruited 18 adults with active SLE who had not previously received any treatment or medical procedures. Age-, sex-, and race-matched healthy donors (without inflammatory diseases) were recruited to participate in the study. These studies were approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (treatment-naïve SLE patients were designated as number 2021-IIT-464), and each participant signed a written informed consent form before enrollment.

[0084] First, the gene expression profile of RAD18 in peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE) was analyzed based on public databases (GSE61635 and GSE72509). High-throughput data were downloaded using the R package GEOquery, and the retrieved gene expression values ​​were input into the limma package for differentially expressed gene analysis. For microarray datasets, if a gene has multiple probes, the most significant probe was used to represent the gene. Genes with significant differential expression were defined as genes / probes with an adjusted p-value < 0.05. The p-value of limma was corrected using the Benjamini-Hochberg method. Compared with healthy controls, the RAD18 mRNA level in patients with SLE was significantly reduced ( Figure 18 a, b). RAD18 mRNA levels (but not RAD21 and RAD51) in patients with systemic lupus erythematosus are compared with... IFNB1 / IFNA mRNA levels were negatively correlated. Figure 18 c, d).

[0085] Protein levels of RAD18 and phosphorylated IRF3 in monocytes were detected by immunoblotting, and real-time PCR was used for detection. IFNB1 / IFNA mRNA levels. Results showed that RAD18 protein levels in monocytes of patients with active systemic lupus erythematosus were significantly lower than in healthy controls, while phosphorylated IRF3 levels and... IFNB1 / IFNA mRNA levels were significantly higher than in healthy controls. Figure 18 Correlation analysis showed that RAD18 protein levels were correlated with phosphorylated IRF3 levels and IFNB1 / IFNA mRNA levels were negatively correlated. Figure 18 d).

[0086] To evaluate the impact of existing treatments on RAD18 expression, 14 adult patients with active systemic lupus erythematosus (SLE) (The First Affiliated Hospital of Zhejiang University) were recruited from July 10 to November 10, 2024, and treated with glucocorticoids, antimalarial drugs, and immunosuppressants. After treatment, only participants with low SLEDAI-2k scores (SLEDAI < 4) were included in the study, and blood samples were collected (this study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine, No. 2024-IIT-887). CD14-positive monocytes were isolated and analyzed. Results showed that after treatment, the levels of phosphorylated IRF3 in patients' monocytes and... IFNB1 / IFNA mRNA levels decreased, but RAD18 protein levels did not change significantly. Figure 18 e.g., after treatment, the negative correlation between RAD18 and p-IRF3 or type I IFNs disappeared. Figure 18 h).

[0087] Further comparisons were made between RAD18 and other E3 ubiquitin ligases (TRIM21, TRIM26, RBCK1, UBE3C) as therapeutic targets for systemic lupus erythematosus (SLE). Using LPS-stimulated human monocyte-macrophages (MDMs) as a model, lentiviruses overexpressing different E3 ubiquitin ligases were constructed and introduced into MDMs. Results showed that enhancing RAD18 expression most significantly inhibited the expression of type I IFNs (including IFNB and IFNA) in MDMs upon LPS stimulation. These results indicate that drugs developed using a strategy of enhancing RAD18 expression achieve better therapeutic effects on SLE compared to drugs targeting other E3 ubiquitin ligases.

[0088] These results indicate that promoting RAD18 expression or activity can inhibit the overexpression of type I interferon, providing a new strategy for the treatment of systemic lupus erythematosus.

[0089] Based on the results of Examples 1-7 above, this invention proposes a complete mechanism model for RAD18 regulation of the IRF3-IFN pathway. Figure 18 i): Upon activation of the RIG-I pathway, IRF3 is phosphorylated and forms a dimer; the p-IRF3 dimer enters the nucleus and reacts with... IFNB1 Promoter binding is required to exert transcriptional activity; RAD18 specifically recognizes and binds to promoters located at [location missing]. IFNB1p-IRF3 on the promoter; RAD18 attaches a K63-linked polyubiquitin chain to p-IRF3 at lysine 193; the ubiquitinated p-IRF3 dimer dissociates from the transcription complex, thereby terminating the transcription. IFNB1 Transcription; ubiquitinated p-IRF3 dimers are exported to the extranuclear space; ubiquitinated p-IRF3 dimers are recognized by OPTN and sent into autophagosomes for degradation.

Claims

1. The application of reagents that regulate RAD18 expression or activity in the preparation of drugs for bidirectional regulation of type I interferon expression, characterized in that, The applications include: enhancing type I interferon expression using reagents that inhibit RAD18 expression or activity; or inhibiting type I interferon expression using reagents that promote RAD18 expression or activity.

2. The application as described in claim 1, characterized in that, The bidirectional regulation is achieved through a RAD18-mediated p-IRF3 ubiquitination degradation mechanism.

3. The application as described in claim 2, characterized in that, The RAD18 phosphorylates IRF3 through K63-type polyubiquitination modification.

4. The application as described in claim 2 or 3, characterized in that, The ubiquitinated p-IRF3 is recognized by the autophagy receptor OPTN and recruited to autophagosomes for degradation, thereby negatively regulating the transcriptional expression of type I interferon.

5. The application as described in any one of claims 1 to 4, characterized in that, The inhibition is achieved by one of the following methods: using an inhibitory nucleic acid molecule, knocking out or knocking down the RAD18 gene, using a CRISPR-Cas9 gene editing system, or using a small molecule compound that blocks the binding of RAD18 to p-IRF3.

6. The application as described in any one of claims 1 to 4, characterized in that, The promotion is achieved by one of the following methods: using a promoting nucleic acid molecule, using a RAD18 overexpression vector, or using a small molecule compound that promotes the binding of RAD18 to p-IRF3.

7. Application of reagents that inhibit RAD18 expression or activity in the preparation of drugs that enhance type I interferon expression to combat RNA virus infection.

8. The application as described in claim 7, characterized in that, The RNA virus is selected from any one or more of the following: SARS-CoV-2, vesicular stomatitis virus, H1N1 influenza virus, or Zika virus.

9. Application of reagents that promote RAD18 expression or activity in the preparation of drugs that reduce the overexpression of type I interferon in patients with systemic lupus erythematosus.

10. The application as described in claim 9, characterized in that, The systemic lupus erythematosus (SLE) patients mentioned were those with active SLE, whose RAD18 protein levels in monocytes were lower than those in healthy controls, and IFNB1 / IFNA mRNA levels were higher than in healthy controls.

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