Use of lapf in the preparation of antiviral drugs

Abstract

The application belongs to the field of biological medicine, and provides application of LAPF in preparation of antiviral drugs. By administering LAPF protein, nucleic acid molecules coding LAPF or functional enhancers thereof, lysosome acidification in dendritic cells or macrophages is enhanced, TLR9 is promoted to transfer to lysosomes and type I interferon pathway is activated. Experimental results prove that by administering LAPF protein, nucleic acid molecules coding LAPF or functional enhancers thereof, DNA virus load such as HSV-1 can be significantly reduced, survival rate of infected mice can be improved, and IFN-alpha / beta production can be enhanced, and good antiviral immune enhancement effect is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of LAPF in the preparation of antiviral drugs. Background Technology

[0002] Lysosomes are important organelles in nucleated cells, participating not only in the degradation and recycling of intracellular substances but also playing a crucial role in innate immunity. As an important component of the intracellular digestive system, lysosomes, through their internal acidic environment and hydrolytic enzymes, can effectively degrade endogenous or exogenous substances, including invading pathogenic microorganisms. The function of lysosomes is particularly important in antiviral immunity. When a virus is endocytosed by a cell, the virus-containing phagosome fuses with the lysosome to form a phagolysosome. Supported by the acidic environment of the lysosome, various hydrolytic enzymes are activated, thereby degrading viral particles. This process is not only a direct pathway for viral clearance but also an important prerequisite for activating immune recognition.

[0003] Toll-like receptor 9 (TLR9) is a pattern recognition receptor located on the lysosomal membrane within cells, capable of specifically recognizing unmethylated CpG motifs in bacterial and viral DNA. TLR9 activation is highly dependent on the acidic environment of the lysosome: under acidic conditions, TLR9 matures after being cleaved by proteases, gaining the ability to recognize DNA; activated TLR9 initiates a downstream signaling cascade, inducing the production of type I interferon (IFN-I), which in turn activates a series of interferon-stimulated genes (ISGs), establishing an antiviral state.

[0004] Lysosomal dysfunction is closely related to a variety of diseases. Impaired lysosomal acidification can lead to impaired TLR9 activation, weakening the body's immune recognition and clearance of DNA viruses, creating conditions for viral latency and reactivation. Herpesviruses such as HSV-1 utilize this mechanism to evade host immune surveillance, establish latent infection in sensory ganglia, and reactivate when the body's immunity declines, causing serious complications such as herpes zoster and postherpetic neuralgia.

[0005] LAPF (also known as PLEKHF1) is a lysosome-associated adaptor protein containing FYVE and PH domains. Located on the lysosomal membrane, it participates in endosome transport and phagosome maturation. Previous studies have shown that LAPF supports bacterial clearance in macrophages by maintaining lysosomal function. However, whether LAPF participates in regulating lysosomal acidification and thus affects TLR9-mediated antiviral immune responses remains unreported. Summary of the Invention

[0006] The first aspect of the present invention provides the use of LAPF in the preparation of antiviral drugs.

[0007] Specifically, the antiviral drug is used to prevent or treat diseases caused by DNA virus infection.

[0008] More specifically, the DNA virus is selected from at least one of herpesvirus, adenovirus, poxvirus, or papillomavirus.

[0009] Optionally, the drug is used to prevent or treat any one or more of the following: postherpetic neuralgia, herpetic encephalitis, herpetic keratitis, neonatal herpes, mechanical hyperalgesia, neuroinflammation, axonal demyelination, and mitochondrial damage.

[0010] More specifically, the drug comprises at least one of the following: a nucleic acid molecule encoding LAPF or its expression vector, LAPF protein or its functional fragment, a compound capable of upregulating LAPF expression levels, and a compound capable of enhancing the biological activity of LAPF.

[0011] A second aspect of the present invention provides an antiviral pharmaceutical composition comprising at least one of a nucleic acid molecule encoding LAPF or its expression vector, LAPF protein or its functional fragment, a compound capable of upregulating LAPF expression levels or a compound capable of enhancing the biological activity of LAPF, and pharmaceutically acceptable excipients.

[0012] Specifically, the dosage forms of the antiviral drug composition include liquid solutions, lyophilized powders, tablets, capsules, granules, sprays, implants, creams, ointments, patches, sustained-release formulations, or suppositories.

[0013] Specifically, the antiviral drug composition can be administered orally, by injection, implantation, external application, spray, inhalation, or a combination thereof.

[0014] More specifically, the injection includes intrathecal injection, intravenous injection, intramuscular injection, or subcutaneous injection.

[0015] A third aspect of the present invention provides a kit for screening candidate drugs that enhance lysosomal acidification and TLR9-mediated antiviral immune responses, the kit comprising the following steps: S1. Contact the candidate drug with cells expressing LAPF; S2. Detect at least one of the following indicators: (i) Degree of lysosomal acidification; (ii) Lysosomal membrane stability; (iii) TLR9 activation level; (iv) Expression levels of type I interferon or its downstream ISGs; (v) HSV-1 viral replication level; S3. Select candidate drugs that can enhance lysosomal acidification, enhance TLR9 activation, upregulate the expression of type I interferon or its downstream ISGs, or inhibit HSV-1 viral replication as potential drugs to enhance lysosomal acidification and TLR9-mediated antiviral immune responses.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention reveals for the first time a novel mechanism by which LAPF enhances the body's innate immunity against DNA viruses by promoting TLR9 activation and type I interferon pathway activation through enhancing lysosomal acidification and lysosomal membrane stability, thus providing a new molecular target for the development of antiviral drugs.

[0017] 2. This invention, by administering LAPF protein, nucleic acid molecules encoding LAPF, or their functional enhancers, can significantly reduce the viral load of DNA viruses such as HSV-1, improve the survival rate of infected mice, and enhance the production of IFN-α / β, exhibiting good antiviral immune enhancement effects, especially in the fight against herpesviruses and related neurological complications, which has important application value. Attached Figure Description

[0018] Figure 1 Hierarchical cluster analysis (A) and principal component analysis (BC) were used to show the clustering and distribution of spinal dorsal horn samples from the PHN models of Lapf-CKO and Lapf-WT mice.

[0019] Figure 2 The expression of several key differentially expressed genes in the dorsal horn of the spinal cord in Lapf-CKO and Lapf-WT mouse PHN models is shown in Figure A, which is a heatmap. Figure B shows the GO enrichment analysis of differentially expressed genes, and Figure C shows the KEGG enrichment analysis of differentially expressed genes.

[0020] Figure 3 The heatmap shows the expression changes of TLR family members (TLR1, TLR3, TLR4, TLR7, TLR8, TLR9) in differentially expressed genes.

[0021] Figure 4 This is a protein-protein interaction network diagram of immune-related differentially expressed genes and TLR9.

[0022] Figure 5 GO enrichment analysis for differentially expressed immune-related genes.

[0023] Figure 6 KEGG enrichment analysis for differentially expressed immune-related genes.

[0024] Figure 7The results show that LAPF enhances microglial innate antiviral immunity by increasing lysosomal acidity and lysosomal membrane stability. A: Confocal microscopy shows changes in lysosomal acidity in BV-2 cells under different treatment groups (control group (Mock+Veh), CQ treatment group (Mock+CQ), Lapf overexpression group (OE-Lapf+Veh), and Lapf overexpression + CQ combined treatment group (OE-Lapf+CQ)) detected by LysoTracker Red staining. The red fluorescence intensity was significantly enhanced in the Lapf overexpression group, and this effect was eliminated by CQ (chloroquine, a lysosomal acidification inhibitor). B: Confocal microscopy shows changes in lysosomal membrane stability in BV-2 cells under different treatment groups detected by AO staining. The orange-red fluorescence intensity was significantly enhanced in the Lapf overexpression group, and this effect was eliminated by CQ treatment. C: Western blot analysis showed the expression level of CTSD (mature form) in BV-2 cells under different treatment groups. The expression of mature CTSD was significantly increased in the Lapf overexpression group, and this effect was eliminated by CQ treatment. DE: Q-PCR was used to detect the transcriptional levels of IFN-α (D) and IFN-β (E) in BV-2 cells after HSV-1 infection in different treatment groups. IFN-I expression was significantly increased in the Lapf overexpression group, and CQ treatment eliminated this effect. FG: Q-PCR was used to detect the transcriptional levels of CXCL10 (F) and ISG15 (G) in BV-2 cells after HSV-1 infection in different treatment groups. ISG expression was significantly increased in the Lapf overexpression group, and CQ treatment eliminated this effect. HI: ELISA was used to detect the protein levels of IFN-α (H) and IFN-β (I) in BV-2 cells after HSV-1 infection in different treatment groups. IFN-I protein level was significantly increased in the Lapf overexpression group, and CQ treatment eliminated this effect. J: Q-PCR was used to detect the expression level of HSV-1 TK DNA in BV-2 cells after HSV-1 infection in different treatment groups. Viral load was significantly reduced in the Lapf overexpression group, and CQ treatment eliminated this effect.

[0025] Figure 8The results showed that LAPF-enhanced lysosomal acidification-mediated antiviral innate immunity is highly dependent on TLR9 activation. AD: Q-PCR was used to detect the transcriptional levels of IFN-α (A), IFN-β (B), CXCL10 (C), and ISG15 (D) in BV-2 cells from different treatment groups (control group, HSV-1 infection group, CpG-C ODN group, and INH-1 ODN group). CpG-C ODN, as a TLR9-specific agonist, mimicked HSV-1 infection-induced IFN-I and ISG expression; INH-1 ODN, as a TLR9-specific inhibitor, significantly inhibited HSV-1 infection-induced IFN-I and ISG expression. EF: ELISA was used to detect the protein levels of IFN-α (E) and IFN-β (F) in BV-2 cells from different treatment groups. The results were consistent with the transcriptional levels, further confirming the effects of CpG-C ODN activating TLR9 and INH-1 ODN inhibiting TLR9. GJ: Q-PCR was used to detect the transcriptional levels of IFN-α (G), IFN-β (H), CXCL10 (I), and ISG15 (J) in BV-2 cells after HSV-1 infection in different treatment groups (control group, Lapf overexpression group, and Lapf overexpression + INH-1 ODN group). Lapf overexpression significantly upregulated the expression of IFN-I and ISGs, while INH-1 ODN treatment completely eliminated this effect of Lapf overexpression, indicating that the antiviral immune-enhancing effect of LAPF depends on TLR9 activation. KL: ELISA was used to detect the protein levels of IFN-α (K) and IFN-β (L) in BV-2 cells after HSV-1 infection in different treatment groups, and the results were consistent with the transcriptional levels. M: Q-PCR was used to detect the expression level of HSV-1 TK DNA in BV-2 cells after HSV-1 infection in different treatment groups. Lapf overexpression can significantly inhibit viral replication, while INH-1 ODN treatment can completely eliminate this effect of Lapf overexpression, further confirming that the antiviral effect of LAPF depends on TLR9 activation.

[0026] Figure 9 The changes in IFN-I, ISGs and HSV-1 viral load in BV-2 cells overexpressing Lapf were detected by Q-PCR and ELISA. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but this should not be construed as limiting the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

[0028] Example 1: Transcriptome sequencing analysis of the dorsal horn of the spinal cord in Lapf-CKO mice 1. Experimental Objective The effects of Lapf microglia-specific knockout on the gene expression profile of the dorsal horn of the spinal cord in PHN mice were analyzed by transcriptome sequencing to explore downstream pathways regulated by LAPF.

[0029] 2. Experimental Methods Microglia-specific LAPF gene knockout mice were constructed using the Cre-LoxP system (Shanghai Southern Model Biotechnology Co., Ltd., n=100 mice). The mice used for short (Lapf-CKO) and their littermate control mice (Lapf-WT) are also used for this purpose.

[0030] PHN mouse model establishment: Hair was removed from the back, sides, and hind limbs of mice, and HSV-1 (1×10⁻⁶) was injected into the anterior part of the right tibia. 6 PFU, 10 μL).

[0031] Total RNA extraction: Spinal dorsal horn tissue was collected from Lapf-CKO and Lapf-WT PHN mice (n=3 / group). Approximately 20 mg of the spinal dorsal horn tissue was added to 1 mL of TRIzol reagent and homogenized thoroughly on ice using a homogenizer. After standing at room temperature for 5 minutes, 200 μL of chloroform was added, and the mixture was vigorously shaken for 15 seconds and then stood at room temperature for 3 minutes. The mixture was centrifuged at 12000g for 15 minutes at 4℃. The upper aqueous phase was transferred to a new EP tube, and an equal volume of isopropanol was added. After mixing, the mixture was stood at room temperature for 10 minutes. After centrifugation at 12000g for 10 minutes at 4℃, the supernatant was discarded. The precipitate was washed twice with 75% ethanol, dried at room temperature, and dissolved in 20 μL of DEPC water. RNA concentration and purity were determined using Nanodrop 2000 (A260 / A280 between 1.9 and 2.1).

[0032] Transcriptome sequencing was performed. Differentially expressed genes (DEGs) were analyzed using the DESeq2 software package, with the selection criteria being |log2FC|≥1 and P<0.05. GO and KEGG enrichment analyses of the DEGs were performed using the clusterProfiler software package.

[0033] 3. Experimental Results Hierarchical cluster analysis (HCA) and principal component analysis (PCA) showed that the Lapf-CKO and Lapf-WT samples clustered into one class, with good homogeneity within each group. Figure 1 AC). The heatmap shows multiple differentially expressed genes between the two groups ( Figure 2 A). GO enrichment analysis showed that DEGs were significantly enriched in biological processes such as DNA replication, glial cell development, Toll-like receptor signaling pathways, and endocytosis. Figure 2 B). KEGG enrichment analysis further confirmed that DEGs were enriched in Toll-like receptor signaling pathways, etc. Figure 2C). Further analysis of TLR family member expression revealed that the expression of TLR1, TLR3, TLR4, TLR7, TLR8, and TLR9 in the dorsal horn of the spinal cord of Lapf-CKO mice was significantly reduced, with TLR9, as a DNA recognition receptor located in lysosomes, showing the most significant downregulation. Figure 3 Protein-protein interaction network analysis revealed that TLR9 interacts with multiple immune-related DEGs and has a positive regulatory effect on most genes. Figure 4 GO and KEGG enrichment analyses of immune-related DEGs revealed their enrichment in IFN-I production, phagocytosis, endocytosis, TLR signaling pathways, and apoptosis. Figure 5 These results indicate that lysosomal and TLR9-mediated antiviral innate immunity is involved in LAPF-mediated PHN progression.

[0034] Example 2 Colocalization analysis of LAPF and lysosomes 1. Experimental Objective To verify the subcellular localization of LAPF in cells and clarify its relationship with lysosomes.

[0035] 2. Experimental Methods BV-2 microglia were cultured and seeded in confocal culture dishes until 70-80% confluence. Cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% goat serum for 1 hour. Rabbit anti-LAPF antibody (1:200) and rat anti-LAMP1 antibody (1:200) were added, and the cells were incubated overnight at 4°C. After washing with PBS, the corresponding fluorescent secondary antibodies (Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 594-labeled goat anti-rat IgG) were added, and the cells were incubated at room temperature in the dark for 1 hour. After washing with PBS, the nuclei were stained with DAPI for 5 minutes, mounted, and observed and photographed under a confocal microscope.

[0036] 3. Experimental Results Confocal microscopy revealed that LAPF (red fluorescence) and the lysosomal marker LAMP1 (green fluorescence) exhibited significant co-localization in the cytoplasm, while Merge images showed a yellow fluorescence signal. Figure 6 This result indicates that LAPF is located in lysosomes, providing a structural basis for LAPF's regulation of lysosomal function.

[0037] Example 3: The regulatory effect of LAPF on lysosomal acidity 1. Experimental Objective To investigate whether LAPF regulates lysosomal acidity and the role of lysosomal acidity in LAPF-enhanced antiviral immunity.

[0038] 2. Experimental Methods BV-2 microglia were divided into four groups: control group (Mock+Veh), CQ treatment group (Mock+CQ), Lapf overexpression group (OE-Lapf+Veh), and Lapf overexpression + CQ combined treatment group (OE-Lapf+CQ). The Lapf overexpression group was transfected with a plasmid to overexpress Lapf; the CQ treatment group was treated with 10 μM chloroquine (a lysosomal acidification inhibitor) for 24 hours; the combined treatment group was first transfected with a Lapf overexpression plasmid, followed by CQ treatment for 24 hours. After treatment, 100 nmol LysoTracker Red was added, and the cells were incubated at 37°C for 30 minutes. After washing with PBS, the cells were observed and photographed under a confocal microscope. The fluorescence intensity of LysoTracker Red reflected lysosomal acidity. Cells from each group were collected simultaneously. Twenty-four hours after HSV-1 infection, cells and culture supernatant were collected. Q-PCR was used to detect the expression of IFN-α, IFN-β, CXCL10, ISG15, and HSV-1 TK DNA. ELISA was performed according to the kit instructions to detect IFN-α and IFN-β protein levels. The kits used in this invention include the IFN-α Mouse ELISA Kit (Invitrogen, BMS6027), the IFN-β Mouse ELISA Kit (Invitrogen, 424001), the TNF-α Mouse ELISA Kit (Invitrogen, 88-7324-88), and the IL-1β Mouse ELISA Kit (Invitrogen, 900-M47).

[0039] Q-PCR method: Take 1 μg of total RNA and reverse transcribe it into cDNA using the PrimeScript RT kit. Reaction system: 4 μL of 5×PrimeScript Buffer, 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of Oligo dT Primer (50 μM), 1 μL of Random 6 mers (100 μM), 1 μg of total RNA, and RNase-free dH2O to 20 μL. Reaction conditions: 37℃ for 15 minutes, 85℃ for 5 seconds, and store at 4℃. Q-PCR detection of HSV-1 TK gene expression: Reaction system: 10 μL of SYBR Premix ExTaq II, 0.8 μL each of forward and reverse primers (10 μM), 0.4 μL of ROX Reference Dye II, 2 μL of cDNA template, 6 μL of dH2O, total system 20 μL. Reaction conditions: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 60℃ annealing and extension for 34 seconds, 40 cycles. Detection was performed using an ABI QuantStudio 5 real-time quantitative PCR instrument. β-actin was used as an internal control, and the relative expression level was calculated using the 2-ΔΔCT method.

[0040] IFN-α primer sequence: Positive 5'-TACTCAGCAGACCTTGAACCT-3' Reverse 5'-CAGTCTTGGCAGCAAGTTGAC-3' IFN-β primer sequence: Positive 5'-ATGAGTGGTGGTTGCAGGC-3' Reverse 5'-TGACCTTTCAAATGCAGTAGATTCA-3' CXCL10 primer sequence: Positive 5'-TCTGAGTGGGACTCAAGGGAT-3' Reverse 5'-GAGGCTCTCTGCTGTCCATC-3' ISG15 primer sequence: Positive 5'-TCTGACTGTGAGAGCAAGCAG-3' Reverse 5'-ACCTTTAGGTCCCAGGCCATT-3' HSV-1 TK primer sequence: Positive 5'-ATACCGACGATATGCGACCT-3'; Reverse 5'-TTATTGCCGTCATAGCGCGG-3'; β-actin primer sequence: Positive 5'-AGAGGGAAATCGTGCGTGAC-3'; Reverse 5'-CAATAGTGATGACCTGGCCGT-3'.

[0041] 3. Experimental Results LysoTracker Red staining results showed that obvious red fluorescent granules were visible in the control group cells, reflecting the normal level of lysosomal acidification; the red fluorescence intensity was significantly enhanced in the Lapf overexpression group, indicating that LAPF overexpression can enhance lysosomal acidification; the red fluorescence in the CQ treatment group almost disappeared, indicating that CQ effectively inhibited lysosomal acidification; the red fluorescence intensity of the Lapf overexpression + CQ combined treatment group was comparable to that of the CQ treatment group alone, indicating that CQ treatment can completely eliminate the effect of LAPF overexpression in enhancing lysosomal acidification. Figure 7 A).

[0042] Functional experiments showed that the mRNA levels of IFN-α, IFN-β, CXCL10, and ISG15 in the Lapf overexpression group were significantly higher than those in the control group, while the HSV-1 TK DNA level was significantly lower than that in the control group. In the CQ treatment group, the mRNA levels of IFN-I and ISGs were significantly lower than those in the control group, while the HSV-1 TK DNA level was significantly higher than that in the control group. The IFN-I and ISGs mRNA levels and HSV-1 TK DNA levels in the Lapf overexpression + CQ combined treatment group were comparable to those in the CQ-only treatment group, indicating that CQ treatment can completely eliminate the antiviral immune-enhancing effect of LAPF overexpression. Figure 7 DG, 7J). ELISA results were consistent with Q-PCR results. Figure 7 (HI). The above results indicate that LAPF promotes antiviral immune responses by enhancing lysosomal acidification.

[0043] Example 4: The regulatory effect of LAPF on lysosomal membrane stability 1. Experimental Objective To investigate whether LAPF regulates lysosomal membrane stability and its role in antiviral immunity.

[0044] 2. Experimental Methods Cell grouping and treatment were the same as in Example 3. After treatment, 5 μM acridine orange (AO) was added, and the cells were incubated at 37°C for 15 minutes. After washing with PBS, the cells were observed and photographed under a confocal microscope. The intensity of orange-red fluorescence reflected the integrity of the lysosomal membrane. Cell proteins were collected simultaneously, and the maturation level of CTSD (cathepsin D) was detected by Western blot.

[0045] 3. Experimental Results AO staining results showed that obvious orange-red fluorescent granules were visible in the control group cells, reflecting normal lysosomal membrane integrity; the orange-red fluorescence intensity was significantly enhanced in the Lapf overexpression group, indicating that LAPF overexpression can enhance lysosomal membrane stability; the orange-red fluorescence in the CQ treatment group was significantly weakened, indicating that CQ treatment led to a decrease in lysosomal membrane stability; the orange-red fluorescence intensity of the Lapf overexpression + CQ combined treatment group was comparable to that of the CQ treatment group alone. Figure 7 B).

[0046] Western blot results showed that a certain level of mature CTSD was observed in the control group; the expression level of mature CTSD was significantly increased in the Lapf overexpression group, indicating that LAPF overexpression promotes the maturation of lysosomal hydrolases; the expression of mature CTSD was significantly decreased in the CQ treatment group; the expression of mature CTSD in the Lapf overexpression + CQ combined treatment group was comparable to that in the CQ alone treatment group. Figure 7 C). The above results indicate that LAPF can enhance lysosomal membrane stability and promote the maturation of lysosomal hydrolases, and this effect depends on lysosomal acidification.

[0047] Example 5: Verification of the role of TLR9 in enhancing antiviral immunity with LAPF 1. Experimental Objective To investigate whether TLR9 is involved in LAPF-enhanced lysosomal acidification-mediated antiviral immune responses.

[0048] 2. Experimental Methods Experiment 1: Verification of the effects of TLR9 agonists and inhibitors BV-2 microglia were divided into four groups: control group, HSV-1 infection group, CpG-C ODN treatment group, and INH-1 ODN treatment group. CpG-C ODN is a TLR9-specific agonist containing a synthetic oligodeoxynucleotide with an unmethylated CpG sequence; INH-1 ODN is a TLR9-specific inhibitor. The HSV-1 infection group was infected with HSV-1 for 24 hours; the CpG-C ODN group was treated with 5 μM CpG-C ODN for 24 hours; and the INH-1 ODN group was pretreated with 5 μM INH-1 ODN for 2 hours before HSV-1 infection for 24 hours. Cells and culture supernatants were collected. The mRNA levels of IFN-α, IFN-β, CXCL10, and ISG15 were detected by Q-PCR, and the protein levels of IFN-α and IFN-β were detected by ELISA.

[0049] Experiment 2: Effect of TLR9 inhibitors on LAPF overexpression BV-2 microglia were divided into three groups: control group (Vector), Lapf overexpression group (OE-Lapf), and Lapf overexpression + INH-1 ODN combined treatment group (OE-Lapf + INH-1 ODN). The Lapf overexpression group was transfected with an overexpressing Lapf plasmid; the combined treatment group was first transfected with a Lapf overexpression plasmid, then pretreated with 5 μM INH-1 ODN for 2 hours after 24 hours, followed by HSV-1 infection for 24 hours. Cells and culture supernatants were collected, and the expression of IFN-α, IFN-β, CXCL10, ISG15, and HSV-1TK DNA was detected by Q-PCR. IFN-α and IFN-β protein levels were detected by ELISA.

[0050] 3. Experimental Results Results of Experiment 1: CpG-C ODN treatment significantly upregulated the mRNA levels of IFN-α, IFN-β, CXCL10, and ISG15, mimicking HSV-1 infection-induced IFN-I and ISG expression; INH-1 ODN treatment significantly inhibited HSV-1 infection-induced IFN-I and ISG expression. Figure 8 AD). ELISA results were consistent with mRNA levels ( Figure 8 The above results indicate that CpG-CODN can act as a TLR9 agonist to activate the TLR9 signaling pathway, while INH-1 ODN can act as a TLR9 inhibitor to block the TLR9 signaling pathway.

[0051] Results of Experiment 2: The mRNA levels of IFN-α, IFN-β, CXCL10, and ISG15 in the Lapf overexpression group were significantly higher than those in the control group, while the HSV-1 TK DNA level was significantly lower than that in the control group, indicating that LAPF overexpression can enhance antiviral immunity and inhibit viral replication; the IFN-I and ISGs mRNA levels and HSV-1 TK DNA levels in the Lapf overexpression + INH-1 ODN combined treatment group were comparable to those in the control group, indicating that INH-1 ODN treatment can completely eliminate the effect of LAPF overexpression in enhancing antiviral immunity. Figure 8 GM). ELISA results were consistent with Q-PCR results. Figure 8 The above results indicate that LAPF-enhanced lysosomal acidification-mediated antiviral innate immunity is highly dependent on TLR9 activation.

[0052] Example 6: Lapf in vivo antiviral experiment The PHN mouse model procedure was the same as in Example 1. LAPF was overexpressed in the mouse spinal cord via intrathecal injection of the AAV vector. Both the AAV2 / 8-LAPF overexpressing virus and the AAV2 / 8 empty vector control virus (AAV-NC) were purchased from Shanghai Heyuan Biotechnology (Group) Co., Ltd., with a viral titer of 1.0 × 10⁻⁶. 12 The vector backbone was pAAV-hSyn-MCS-EGFP, with a concentration of vg / mL. On day 7 after animal modeling, mice underwent intrathecal AAV virus injection, with 5 μL of AAV virus stock solution slowly injected intrathecally into each mouse. The control group received an equal volume of empty AAV2 / 8-NC virus simultaneously. After 14 days of feeding following virus injection, once the target gene LAPF was stably overexpressed, spinal cord dorsal horn tissue was collected to detect the expression of IFN-I, ISGs, inflammatory factors, neuroprotective factors, and HSV-1 viral load. The detection methods were the same as in Example 3, and the experimental results are shown below. Figure 9 The control group was the AAV-NC group, and the LAPF overexpression group was the AAV-NC group. Lapf Group.

[0053] Overexpression of LAPF significantly increased the transcriptional levels of IFN-I and ISGs, as well as the IFN-I protein level, while reducing HSV-1 viral load and significantly decreasing the transcriptional and protein levels of inflammatory factors TNF-α and IL-1β.

[0054] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of LAPF in the preparation of antiviral drugs, characterized in that, The antiviral drug is used to prevent diseases caused by DNA virus infection; the DNA virus is HSV-1 virus; the drug is a nucleic acid molecule encoding LAPF or its expression vector.

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