Application of deacetylase Sirt4 and medicine comprising deacetylase Sirt4 expression inhibitor

Abstract

The invention belongs to the technical field of antiviral drug research, and particularly relates to application of deacetylase Sirt4 and a drug comprising a deacetylase Sirt4 expression inhibitor. It is found through experiments that Sirt4 can be induced by HSV-1 infection, and the antiviral innate immune response triggered by HSV-1 can be actively adjusted. The knockdown or knockout of the Sirt4 obviously inhibits the excessive activation of the anti-DNA virus innate immune response.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral drug research, and specifically relates to an application of a deacetylase Sirt4 and a drug comprising a deacetylase Sirt4 expression inhibitor. Background Art

[0002] The appearance of DNA in the cytoplasm is a potential danger signal, which usually means that the invasion of pathogenic microorganisms has caused the occurrence of infection or tissue damage, and can quickly trigger the body's innate immune response. The innate immune response triggered by cytoplasmic DNA depends on the recognition of cytoplasmic DNA by DNA recognition receptors and is an important part of the body's defense. However, excessive or sustained activation of the antiviral innate immune response can also cause damage to the body's organs or tissues. For example, excessive activation of the antiviral innate immune response can cause a large number of cytokines (such as IL-6, TNF-α, etc.) and inflammatory mediators to be released sharply in a short period of time, forming a "cytokine storm". This violent inflammatory response can damage the blood vessel wall, cause tissue damage, organ dysfunction, and even endanger life. For another example, the inflammatory response caused by the sustained antiviral innate immune response will not only directly damage the tissues at the site of infection, but may also affect organs far away from the site of infection, such as the lungs, heart, and kidneys, through systemic reactions, leading to multiple organ dysfunction syndrome (MODS). In addition, excessive immune response may also induce autoimmune diseases, further exacerbating the condition. Therefore, the intensity and duration of activation of the antiviral innate immune response need to be strictly and precisely regulated to avoid causing harm to the body.

[0003] Cyclic GMP-AMP synthase (cGAS) is a key DNA recognition receptor that plays an important role in both DNA virus and retrovirus infection. Although there are many cytoplasmic DNA recognition receptors involved in the innate immune signaling pathway induced by viral DNA, cGAS is the most common DNA recognition receptor in most cells based on current research. It forms "cGAS-DNA" droplets during viral infection and produces cGAMP. As a second messenger, cGAMP binds to and mediates the activation of the stimulator of interferon genes (STING, also known as MITA / MPYS / ERIS), thereby inducing the activation of downstream TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), thereby producing type I interferon and subsequent antiviral immune response. In recent years, studies have shown that cGAS functions as a major DNA recognition receptor in the process of exogenous DNA transfection, bacterial infection, DNA virus or retrovirus infection, and leakage of self-DNA such as mitochondrial DNA and chromosomal DNA. The acetylation modification of cGAS is very important for regulating its function, but the regulation of cGAS function by deacetylase is still unclear.

[0004] Sirt4 is a member of the important deacetylase family and is a NAD + Sirt4 is a deacetylase that plays an important role in cell metabolism. In the human body, Sirt4 is a key regulatory molecule in a variety of mitochondrial metabolic processes. It has been shown to play a vital role in fatty acid and branched-chain amino acid metabolism, tricarboxylic acid cycle, electron transfer, reactive oxygen production, insulin secretion and sensitivity, cell apoptosis, ATP homeostasis, redox state, etc. In addition, Sirt4 also has the function of inhibiting tumor growth. However, there is no report on the correlation between Sirt4 and antiviral infection. Summary of the invention

[0005] In view of the above technical problems, the present invention provides an application of a deacetylase Sirt4 as a drug target for screening drugs for preventing, alleviating or treating HSV-1 virus infection.

[0006] The specific technical solutions provided by the present invention are as follows:

[0007] In a first aspect, the present invention provides an application of a deacetylase Sirt4 as a drug target for screening drugs for preventing, alleviating or treating HSV-1 viral infection, wherein the application is used for non-disease diagnosis or treatment.

[0008] In a second aspect, the present invention provides a method for screening drugs for preventing, alleviating or treating HSV-1 virus infection using the deacetylase Sirt4, comprising the following steps:

[0009] The candidate drug is administered to the HSV-1 virus infection model, and the expression level of the deacetylase Sirt4 before and after administration is detected. The expression level of the deacetylase Sirt4 after administration is lower than the expression level of the deacetylase Sirt4 before administration, which indicates that the candidate drug is a target drug.

[0010] In a third aspect, the present invention provides a drug for regulating HSV-1 virus infection obtained by screening according to the above method, wherein the drug comprises an expression inhibitor of deacetylase Sirt4, wherein the expression inhibitor of deacetylase Sirt4 is siRNA S2 or siRNA S3,

[0011] The sense strand sequence of the siRNA S2 is 5'-CAUCCAGCAUGGUGAUUUU-3', and the antisense strand sequence is 5'-AAAAUCACCAUGCUGGAUG-3';

[0012] The sense strand sequence of the siRNA S3 is 5'-GCGUGUCUGAAACUGAAUU-3', and the antisense strand sequence is 5'-AAUUCAGUUUCAGACACGC-3'.

[0013] Preferably, the drug is used to improve multiple organ dysfunction syndrome caused by excessive activation of antiviral innate immune response.

[0014] Preferably, the drug is used to improve autoimmune diseases caused by excessive activation of antiviral innate immune response.

[0015] Preferably, the drug comprises a pharmaceutically acceptable excipient or carrier.

[0016] Preferably, the drug is prepared as an oral preparation or an injection preparation.

[0017] Preferably, the oral preparation is a granule, capsule, tablet or liquid preparation. The injection preparation can be administered by intramuscular injection or intravenous injection. During the specific preparation, those skilled in the art can select suitable adjuvants or carriers according to the specific dosage form to be prepared. Such as diluents, suspending agents, emulsifiers, surfactants, preservatives, pH regulators, flavoring agents, coating agents, viscosity enhancers, etc.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] Transitional activation of antiviral responses can cause damage to the body, so it is necessary to limit excessive activation of antiviral responses. The present invention found that Sirt4 can be induced by HSV-1 infection and actively regulate the antiviral innate immune response triggered by HSV-1. Knocking down or knocking out Sirt4 significantly inhibited the anti-DNA virus innate immune response, and the ability of Sirt4-deficient mice to resist HSV-1 viral infection was also weakened. Mechanistically, Sirt4 interacts with cGAS and inhibits the acetylation of cGAS, thereby playing a role in positively regulating the anti-DNA virus innate immune response. Therefore, Sirt4 can be used as a target substance for screening drugs for preventing, alleviating or treating HSV-1 viral infection. At the same time, the expression inhibitor of Sirt4 can flexibly adjust the intensity of the antiviral innate immune response, thereby reducing unnecessary tissue damage while ensuring sufficient antiviral ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Sirt4 interacts with cGAS; (A) PMA-THP1 cells were infected with HSV-1 (MOI = 1) for the indicated time, then treated with NAM (5 mM) for 6 h, and subjected to Co-IP and Western Bolt analysis. (B and C) HEK293T cells were transfected with different plasmid combinations as indicated for 24 h, and then subjected to Co-IP and Western Bolt analysis as indicated. (D) PMA-THP1 cells were transfected with HSV60 (1 μg / mL) for the indicated time, and then subjected to Co-IP and Western Bolt analysis as indicated. (E) HeLa cells were treated or not with HSV-1 (MOI = 1) for 8 h, and laser confocal microscopy was performed using Sirt4 (green) antibody and cGAS antibody (red). (F) MEF cells were transfected with Flag-Sirt4 for 24 h, and then treated with HSV-1 (MOI = 1), and then subjected to in situ PLA assay to detect the colocalization of Sirt4 and cGAS. Sirt4-cGAS complex is shown in red; cell nuclei are shown in blue. (G) Schematic diagram of full-length and truncated cGAS (top). HEK293T cells were co-transfected with full-length Flag-cGAS or its truncated form and HA-Sirt4 plasmid, and Co-IP and Western Bolt analysis were performed 24 hours later. (H) HEK293T cells were transfected with various plasmid combinations as indicated, and Co-IP and Western Bolt analysis were performed 24 hours later.

[0021] Figure 2Sirt4 knockdown attenuates the innate immune response induced by HSV-1 or exogenous DNA; (A) PMA-THP1 cells were infected with HSV-1 (MOI=1) or transfected with HSV60 (1μg / mL) for the indicated time, and then Western Bolt analysis was performed. (B) PMA-THP1 cells were transfected with Sirt4 siRNA (S1, S2, and S3) or control (SC) for Western Bolt analysis. (C) PMA-THP1 cells were transfected with Sirt4 siRNA (S2 and S3) or control (SC), and then infected with HSV-1 (MOI=1) for 24h, and the HSV-1 titer was detected by standard plaque assay. (D) PMA-THP1 cells were transfected with Sirt4 siRNA (S2) or control (SC), and then infected with HSV-1 (MOI=1) for 8h, and the cells were lysed for Real-time PCR analysis. (E) PMA-THP1 cells were transfected with Sirt4 siRNA (S2 and S3) or control (SC), and then infected with HSV-1 (MOI = 1) for 24 h for ELISA. (F) PMA-THP1 cells were transfected with Sirt4 siRNA (S2 and S3) or control (SC), and then infected with HSV-1 (MOI = 1) for 8 h, and the cells were lysed for Native-PAGE or SDS-PAGE. (G) PMA-THP1 cells were transfected with Sirt4 siRNA (S2) or control (SC), and then infected with HSV-1 (MOI = 1) for the indicated time, and the cytoplasmic and nuclear fractions were separated and subjected to immunoblotting. * p<0.05, ** p<0.01, *** p<0.001.

[0022] Figure 3 Sirt4 deficiency attenuates DNA virus-induced innate immune responses in BMDM cells; (A) WT and Sirt4 KO BMDM cells were infected with HSV-1 (MOI = 1) for 24 h, and the supernatants were collected for ELISA. (B) WT and Sirt4 KO BMDM cells were infected with HSV-1 (MOI = 1) for the indicated time, and then the cells were lysed for Real-time PCR analysis. (C and D) BMDM cells were infected with HSV-1 (MOI = 1) (C) or transfected with HSV60 (1 μg / mL) (D) for 0, 4, and 8 h, and then subjected to Western Bolt analysis. * p<0.05, ** p<0.01, *** p<0.001.

[0023] Figure 4 Sirt4 deficiency weakens the innate immune response induced by DNA viruses in MEF cells; (A) WT and Sirt4 KO MEF cells were infected with HSV-1 (MOI=1) for 0, 4, and 8 h, and then the cells were lysed for Real-time PCR analysis. (B) WT and Sirt4 KO MEF cells were infected with HSV-1 (MOI=1) for 0, 4, and 8 h, and then Western Bolt analysis was performed. (C) MEF cells were infected with HSV-1 or VSV (MOI=1) for 24 h, and the supernatant was collected for ELISA analysis. * p<0.05, ** p<0.01, *** p<0.001.

[0024] Figure 5 Sirt4 protects mice against HSV-1 infection; (A) Sex- and age-matched wild-type (WT) and Sirt4-deficient (KO) mice (n=4, 8 weeks old) were intravenously infected with HSV-1 (1×10 7 PFU). Body weight changes were monitored for 11 days. (B) Wild-type (WT) and Sirt4-deficient (KO) mice (n=5, 8 weeks old) were intravenously infected with HSV-1 (2×10 7 PFU). The mice were monitored for survival for 15 days. (C) ELISA was used to detect the expression of HSV-1 (1×10 7 PFU) were used to measure the levels of IFN-β, IL-6 and TNF-ɑ in serum 6 h after immunization. (D) Wild-type (WT) and Sirt4-deficient (KO) mice (n=3, 8 weeks old) were infected with HSV-1 (1×10 7 PFU) for 24 hours, and bronchoalveolar lavage fluid (BALF) was collected and tested by ELISA. The data in Figures A and C to D were tested using unpaired t-tests, and the data in Figure B were tested using log-rank tests. * p<0.05, ** p<0.01, *** p<0.001.

[0025] Figure 6 Sirt4 protects mice against HSV-1 infection; (A) Wild-type (WT) and Sirt4-deficient (KO) mice (n=3, 8 weeks old) were intravenously infected with HSV-1 (1×10 7PFU) for 4 days, and then the brains of mice were tested by real-time fluorescence quantitative PCR. (B) Wild-type (WT) and Sirt4-deficient (KO) mice were intravenously infected with HSV-1 (1×10 7 PFU) for 24 h, and then the lungs, livers, and spleens of the mice were tested by ELISA. (C) Wild-type (WT) and Sirt4-deficient (KO) mice were intravenously infected with HSV-1 (1×10 7 PFU) for 24 h, and then the lungs, livers and spleens of the mice were tested by real-time fluorescence quantitative PCR. (D) Sex- and age-matched wild-type (WT) and Sirt4-deficient (KO) mice were intravenously infected with HSV-1 (1×10 7 PFU) for 24 h, and lung sections were analyzed by HE staining. Scale bar, 100 μm. Data in A to C were tested using unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001.

[0026] Figure 7 Sirt4 reduces cGAS acetylation; (A) HEK293T cells were transfected with the indicated plasmids for 24 h, and Co-IP and Western Bolt analysis were performed. (B) HeLaWT and HeLacGAS KO cells were transfected with Sirt4 siRNA (S2) or control (SC) for 24 h, and then infected or not infected with HSV-1 (MOI=1) for 8 h. Western Bolt analysis was performed. (C and D) HEK293T cells were transfected with the indicated plasmids for 24 h, and Co-IP and Western Bolt analysis were performed. (E) PMA-THP1 cells were transfected with Sirt4 siRNA (S2) or control (SC), and then infected with HSV-1 (MOI=1) for 0, 4, and 8 h, and then the cells were lysed for Co-IP and Western Bolt analysis. (F) PMA-THP1 WT and Sirt4 KO cells were infected with HSV-1 (MOI = 1) for 0, 4, and 8 h, and then the cells were subjected to Co-IP and Western Bolt analysis. (G) PMA-THP1 WT and Sirt4 KO cells were stimulated with Biotin-ISD (upper) or Biotin-HSV60 (lower) for 0, 4, and 8 h, and then Co-IP and Western Bolt analysis were performed. * p<0.05, ** p<0.01, *** p<0.001. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below with reference to specific embodiments, but is not limited thereto.

[0028] The present invention provides an application of deacetylase Sirt4 as a drug target for screening drugs for preventing, alleviating or treating HSV-1 virus infection, and the application is used for non-disease diagnosis or treatment.

[0029] The present invention found that Sirt4 can be induced by HSV-1 infection and actively regulates the antiviral innate immune response triggered by HSV-1. Knocking down or knocking out Sirt4 significantly inhibited the anti-DNA virus innate immune response, and the ability of Sirt4-deficient mice to resist HSV-1 viral infection was also weakened. Therefore, Sirt4 can be used as a target substance for screening drugs for preventing, alleviating or treating HSV-1 viral infection.

[0030] 1. Experimental Materials

[0031] 1.1 Research subjects

[0032] Cells: HeLa, cGAS-KO HeLa, THP1, Sirt4-KO THP1, BMDM, Sirt4-KO BMDM, MEF, Sirt4-KO MEF, HEK293T.

[0033] Animals: C57BL / 6 mice; Sirt4 knockout mice were provided by Professor Liang Yinming of the School of Medical Technology, Xinxiang Medical College. All animal experiments were performed in accordance with the guidelines approved by the Animal Care Committee of our school (Ethics Review Approval No.: XYLL-2020166). Age- and sex-matched wild-type and Sirt4-deficient C57BL / 6 mice were used in the experiments.

[0034] 1.2 Main Reagents

[0035] 1.2.1 Reagents used in cell experiments

[0036] Fetal bovine serum, DMEM, VivaCell; RPMI Medium 1640 basic, Gibco; penicillin-streptomycin mixture (100×) for cell culture, Solebol; trypsin digestion solution (phenol red-free), Proteintech; Opti-MEM, Gibco; Lipofectamine 2000, Invitrogen; phorbol myristic acid acetate (PMA), Beyotime; HSV60, InvivoGen; nicotinamide (NAM), Selleck.

[0037] 1.2.2 Reagents used in immunoblotting experiments

[0038] Tris-base, sodium dodecyl sulfate, 30% Acr-Bis (29:1) gel preparation solution, glycine, bromophenol blue, Solebol; tetramethylethylenediamine, Bio-Technology; β-Mercaptoethanol, Biotechnology Grade;

[0039] PageRulerPrestained Protein Ladder, Thermo Fisher; Nitrocellulose membrane (0.45μm), Merck Millipore; Enhanced ECL Chemiluminescent Substrate Kit, Yeasen Biotechnology; Acetylated-Lysine antibody, Phospho-TBK1 antibody, Phospho-IRF3 (Ser) antibody, Phospho-p65 antibody, Sirt4 antibody, Cell Signaling Technology; TBK1 antibody, Flarebio; IRF3 antibody, p65 antibody, β-actin antibody, cGAS antibody, Proteintech; HA antibody, BioLegend; Myc antibody, CUSABIO; Flag antibody, Sigma-Aldrich; Tubulin antibody, Santa Cruz Biotechnology; H3 antibody, Abcam.

[0040] 1.2.3 Reagents used in immunoconfocal experiments

[0041] Bovine serum albumin powder, 4% tissue cell fixative, DAPI solution, NP-40, bovine serum albumin powder, Solebao; TritonX-100, Baosight Biotechnology Co., Ltd.; CoraLite488-Anti-Mouse IgG antibody, Cy3-Anti-Rabbit IgG (H+L) antibody, Proteintech; Protein A / G Agarose, Bio-Tech; Na3VO4, MPBiomedicals.

[0042] 1.2.4 Reagents used in PLA experiments

[0043] 4% tissue cell fixative, bovine serum albumin powder, Solebol; TritonX-100, Baosight Biotechnology Co., Ltd.; Duolink in situ PLA fluorescence detection wash buffer, Duolink in situ PLA probe anti-rabbit PLUS, Duolink in situ PLA probe anti-mouse MINUS, Duolink in situ PLA red detection reagent, Duolink in situ PLA detection mounting medium (containing DAPI), Sigma-Aldrich.

[0044] 1.2.5 Reagents used to construct plasmids

[0045] 2× Prime StarMax Premix, TaKaRa; DNA Marker, Bolkin; Super Red, Gaide Chemical; T4 DNA ligase, Promega.

[0046] 2. Experimental methods

[0047] 2.1 Cell culture

[0048] The revived cells are subcultured and then transfected. The transfection process is as follows:

[0049] (1) When the cell density reaches 70-90% and the cells are in good condition, cell transfection is performed.

[0050] (2) Prepare the transfection system: The total amount of plasmid transfected in each well varies depending on the specifications of the cell plate. Take a 12-well plate as an example: prepare two EP tubes and add 100 μL of Opti-MEM culture medium to each EP tube. Add 2 μL of Lipo2000 to the first EP tube, mix well and let stand for 5 minutes. Add 2 μg of plasmid to the second EP tube and mix well. After 5 minutes, mix the liquids in the two EP tubes and let stand at room temperature for 20 minutes.

[0051] (3) During the static period, discard the original cell culture medium and add 400 μL Opti-MEM culture medium. After the static period, add the plasmid and Lipo2000 mixture to the corresponding cell culture wells, mix well, and after culturing for 4 to 6 hours, discard the transfection mixture and replace it with complete culture medium containing 10% serum.

[0052] 2.2 Immunoblotting

[0053] 2.1.1 Electrophoresis

[0054] Adjust the voltage and time: 200 V, 50 min, and start electrophoresis.

[0055] 2.1.2. Transfer

[0056] Prepare the transfer solution in advance and pre-cool it. After the electrophoresis is completed, carefully open the electrophoresis tank cover, remove the gel sheet from the electrophoresis tank, rinse it slowly with clean water, pry the gel sheet open with a plastic spatula and cut off the excess gel on the upper layer, gently place the remaining gel on the transfer clamp filter paper, soak the nitrocellulose membrane (NC membrane) in the transfer solution and cover the upper layer of the gel, cover the filter paper and filter screen in turn after the bubbles are discharged, clamp the transfer clamp and place it in the transfer tank, pour the pre-cooled transfer solution into the transfer tank until the liquid overflows, and cover the transfer tank cover according to the direction of the electrode. In a low temperature environment, perform electrophoresis at a current of 300mA for about 90 minutes.

[0057] 2.1.3. Closure

[0058] Prepare the blocking solution (5% skim milk) in advance. After the transfer is completed, open the cover of the transfer tank, slowly take out and open the transfer clip, use tweezers to take out the NC membrane in the clip and put it into the blocking solution, and block it at low speed on a shaker at room temperature for 1 hour.

[0059] 2.1.4. Primary Antibody Incubation

[0060] Prepare 1×TBST membrane washing solution in advance. After blocking, pour an appropriate amount of membrane washing solution into the membrane washing box, take out the blocked NC membrane, and put it into the membrane washing solution for washing. Then cut out the NC membrane strips of the corresponding molecular size according to the experimental requirements, use the blocking solution to dilute and prepare the primary antibody in proportion, and then put the NC membrane strips into the antibody box containing the primary antibody, and incubate at room temperature for 2 hours or incubate overnight at 4°C.

[0061] 2.1.5. Secondary Antibody Incubation

[0062] Pour an appropriate amount of membrane washing solution into the membrane washing box, take out the NC membrane strips from the antibody box and put them into the membrane washing box, then put the membrane washing box on a shaker at room temperature, adjust to medium speed, and wash for 8 minutes each time. After the washing time is up, pour out the original membrane washing solution, add an appropriate amount of new membrane washing solution, continue to wash on the shaker for 8 minutes, and repeat the washing three times. During the washing period, use the blocking solution to dilute the secondary antibody in proportion. After washing, put the NC membrane strips into the antibody box containing the secondary antibody and incubate at room temperature for 1 hour.

[0063] 2.2.6. Development

[0064] Take out the NC membrane strips and place them in a membrane washing box containing an appropriate amount of membrane washing solution. Adjust the shaker to medium speed, put on the membrane washing box, and wash 3 times. The washing process is the same as the previous step. Take luminescent liquid A and luminescent liquid B, prepare them in a light-proof EP tube at a volume ratio of 1:1 and mix them with a pipette gun. Turn on the developer, spread the NC membrane strips flat on the developer tray, then evenly cover the NC membrane strips with the luminescent liquid, and then immediately put the developer tray into the developer for development. After development, save the development results. After the development is completed, remove the NC membrane strips, wipe the developer tray, and put the developer tray back into the developer to turn off the developer.

[0065] 2.2 Real-time fluorescence quantitative PCR

[0066] 2.3.1. Extraction of RNA

[0067] The experiment involves tissue RNA extraction and cell RNA extraction. Here we take cell RNA extraction as an example:

[0068] (1) Remove the original culture medium in the cell culture plate. Add 1 mL of RNAiso Plus to each well and repeatedly blow the bottom of the cell culture plate with a pipette to completely lyse the cells. Then transfer all of them to the corresponding RNase-free EP tube.

[0069] (2) After the cells are lysed, 200 μL of pre-cooled chloroform is added to the cells, and then the cells are shaken vigorously using a vortexer to ensure complete fusion, and then placed in an ice box for 10 min.

[0070] (3) Centrifuge the sample at 4°C and 12,000 rpm for 15 min. After centrifugation, aspirate the upper layer into a new RNase-free EP tube.

[0071] (4) Add isopropanol of the same volume as the upper liquid into the EP tube and mix until the filaments in the EP tube disappear completely (if the mixing is not sufficient, the RNA concentration will be affected), and then place it on ice for 10 minutes.

[0072] (5) Centrifuge the sample at 4°C and 12000 rpm for 20 min. After centrifugation, discard the upper liquid, add 600 μL of 75% ethanol solution to each EP tube, mix well, and centrifuge at 4°C and 7500 rpm for 5 min.

[0073] (6) After centrifugation, discard the supernatant and allow the precipitate to dry naturally. The white precipitate will gradually turn transparent. Add an appropriate amount of RNase-free ddH2O (approximately 20 μL) according to the size of the precipitate. Finally, use an ultra-micro spectrophotometer to measure the concentration and purity of the extracted RNA.

[0074] 2.3.2 Synthesis of cDNA

[0075] cDNA synthesis system: template RNA 1μg, Enzyme Mix 1μL, 5×All-in-one qRT SuperMix 4μL, RNase-free ddH2O to a total system of 20μL.

[0076] According to the amount and concentration of template RNA, calculate the volume of template RNA. Use HiScriptIII All-in-one RT SuperMix Perfect for qPCR Kit (Vazyme). Prepare the reaction system in an RNase-free EP tube according to the components in Table 1. Mix well and centrifuge for later use.

[0077] Reverse transcription reaction program: 50°C for 15 min, 85°C for 5 s.

[0078] After reverse transcription, dilute each sample with 20 μL of RNase-free ddH2O.

[0079] 2.3.3 qPCR reaction

[0080] qPCR reaction system: SYBR 5μL, ddH2O 3μL, Primer sp 0.5μL, Primer as 0.5μL.

[0081] 2.3.4 Enzyme-linked immunosorbent assay

[0082] ELISA kit was purchased from Invitrogen:

[0083] (1) Coating: Calculate the number of wells to be coated according to the experimental requirements, prepare a transparent microplate, and calculate the total volume of coating solution required based on 100 μL per well. Dilute the coating solution to 1× in proportion, and use coating buffer to dilute the capture antibody in proportion. After thorough mixing, add 100 μL of coating solution to each well of the microplate. Seal the microplate with a sealing film and place it in a 4°C refrigerator overnight.

[0084] (2) Blocking: Wash each well with about 300 μL of PBST solution (phosphate buffered saline + 0.05% Tween) three times, each wash for 1 min. Calculate the volume of Elisa spot solution required for the experiment, then dilute the Elisa spot solution to 1× in proportion, add 200 μL to each well of the polystyrene microplate for blocking, and incubate at room temperature for 1 h.

[0085] (3) Sample addition: Dilute the standard with 1× Elisa spot solution into four gradients (500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL). Dilute the test sample according to the experimental requirements. Wash each well with about 300μL of PBST solution at least once, each time for 1min. Add the standard and test sample to the corresponding reaction wells in turn, 100μL per well, and incubate at room temperature for 2h.

[0086] (4) Add detection antibody: Wash each well three times with about 300 μL of PBST solution, each wash for 1 min. Calculate the required volume of detection antibody according to the experimental requirements, dilute the detection antibody in proportion with 1× Elisa spot solution, use a pipette to draw up the detection antibody and add it to the corresponding reaction wells in sequence, 100 μL per well, and incubate at room temperature for 1 h.

[0087] (5) Add horseradish peroxidase (HRP): Wash each well three times with about 300 μL of PBST solution, each wash for 1 min. Calculate the required volume of HRP according to the experimental requirements, dilute it proportionally using 1× Elisa spot, use a pipette to draw HRP and add it to the corresponding reaction wells in sequence, 100 μL per well, and incubate at room temperature for 30 min.

[0088] (6) Add TMB substrate solution for color development: Equilibrate the TMB solution to room temperature in advance, and wash each well with about 300 μL of PBST solution 5 times, 1 min each time. Use a pipette to draw up the TMB solution and add it to the reaction wells in sequence, 100 μL per well. Place in a 37°C incubator in the dark for 15 min. The reaction time can be adjusted appropriately according to the color development effect.

[0089] (7) Termination: Add 50 μL / well of stop solution in the order of color development and sample addition.

[0090] (8) Result determination: Turn on the microplate reader, set the wavelength to 450 nm, place the reaction plate in the plate and start reading. Save the reading results, simulate the standard curve based on the absorbance value of the standard, and perform data analysis.

[0091] 2.4 Virus Experiment

[0092] 2.4.1. HSV-1 infection of cells

[0093] According to the experimental dosage and virus titer, HSV-1 virus solution (MOI = 1) was prepared using serum-free DMEM medium. The original cell culture medium in the culture plate was discarded, and sterile PBS was added at 300 μL / well (24-well plate), and the cell culture plate was washed twice. The HSV-1 virus solution was added, and after culturing for 2 hours, the original virus solution in the culture plate was discarded, and the culture plate was washed twice with PBS, and complete culture medium was added to the cell culture plate (24-well plate) at 500 μL / well, and then placed in a cell culture incubator for culture.

[0094] 2.4.2 Virus plaque assay

[0095] (1) According to experimental requirements, Vero cells were plated in a 24-well plate. After overnight, the virus titer could be measured when the Vero cells were fully grown.

[0096] (2) About 24 hours after the cells were infected with the virus, the cell supernatant was collected and diluted in series: prepare three EP tubes, marked as No. 1, No. 2, and No. 3, add 450 μL of serum-free DMEM medium to each tube, pipette 50 μL of cell supernatant into tube No. 1, mix thoroughly, then pipette 50 μL of the mixture from tube No. 1 into tube No. 2, mix thoroughly, then pipette 50 μL of the mixture from tube No. 2 into tube No. 3, mix thoroughly and set aside.

[0097] (3) Discard the original cell culture medium and wash the culture plate twice with PBS. Take 200 μL of the mixture from the above three EP tubes and add it to the cell culture wells. Make two replicates for each dilution. Put it in the incubator for 2 hours, discard the supernatant, wash it twice with PBS, mix the 2×1640 solution and 1.6% agarose gel in equal proportions, add 2% serum and 1% levofloxacin, mix thoroughly, and add the mixture to each well of the cell culture plate at 500 μL / well. After the gel mixture cools and solidifies, turn the cell culture plate upside down and place it in the cell culture incubator for about 72 hours, then take it out, add 1‰ crystal violet-formaldehyde fixative to the cell culture plate, 200 μL / well, leave it at room temperature for 2 hours, tap the gel in the culture plate, count the number of plaques and record the analysis.

[0098] 2.4.3. Amplification of HSV-1

[0099] Discard the original Vero cell culture medium, add 2 / 3mL phosphate buffer to wash the bottom of the cell culture bottle twice, then add an appropriate amount of HSV-1 virus solution and 12mL fresh DMEM culture medium according to the virus titer, mix well, culture for 2 hours, discard the mixed solution, replace it with DMEM culture medium containing 3% serum, and then add levofloxacin at a ratio of 1:100, mix well and culture for 36-48 hours. When the cells are fused and large pieces fall off, centrifuge the cell suspension at 2000rpm for 20 minutes, and collect the supernatant for later use.

[0100] 2.5. HSV-1 Concentration

[0101] Take the amplified virus liquid, centrifuge it at 4°C and 2000rpm for 20min, take the supernatant and filter it with a 0.45μm filter, centrifuge it at 4°C and 3600rpm for 20-30min, stop centrifugation when the upper liquid of the ultrafiltration tube is less than 1mL, collect the virus liquid in a 15mL centrifuge tube, repeat the operation, centrifuge the collected virus liquid at 4°C and 3600rpm for 20-30min, concentrate it to 2-3mL, dispense it into EP tubes, 400μL / tube, and store it at -80°C.

[0102] 2.6. Immunoconfocal Immunoassay

[0103] (1) Discard the cell culture medium, wash with phosphate buffer, add 4% tissue cell fixative to fix the cells, and finally wash with phosphate buffer.

[0104] (2) Add 400 μL of 0.5% TritonX-100 to each well to permeabilize the membrane for 5 min, then wash with 400 μL of 1% BSA solution for 3 times, 1 min each time. During the third wash, retain the supernatant, remove the cell slide from the cell culture well and place it on a plate, immerse the cell slide with 1% BSA solution, and block for 30 min.

[0105] (3) Use 1% BSA solution to dilute the primary antibody at a ratio of 1:100 and mix well for use. After blocking, aspirate and discard the blocking solution on the cell slide, add 50 μL of the primary antibody dilution solution to each cell slide, and incubate at 4°C overnight or at room temperature for 2 hours.

[0106] (4) Select rabbit / mouse fluorescent secondary antibody according to experimental requirements, and dilute the fluorescent secondary antibody with 1% BSA solution according to the antibody instructions. Discard the original primary antibody solution and wash the cell slides 3 times with 1% BSA solution, each wash for 1 min. Add 50 μL of the diluted fluorescent secondary antibody solution to each cell slide and incubate at room temperature in the dark for 1 h.

[0107] (5) Discard the original secondary antibody solution on the cell slide, wash the cell slide 3 times with 1% BSA solution, each wash for 1 min. Add 100 μL of DAPI solution to each cell slide and stain for 10 min. After staining, wash the cell slide 5 times with phosphate buffer.

[0108] (6) Pipette 5-7 μL of sealing agent and drop it on the glass slide. Then use tweezers to hold the cell slide and slowly buckle it on the glass slide. Use nail polish to seal the edge of the cell slide. After the nail polish dries, you can take immunofluorescence confocal microscopy photos or store them in a 4°C refrigerator away from light.

[0109] 2.7 PLA fluorescence experiment

[0110] Duolink in situ PLA fluorescence detection kit was purchased from Sigma-Aldrich

[0111] (1) Preparation of reagents: Dissolve Duolink in situ PLA fluorescence detection wash buffer A powder and B powder in high-purity water, adjust the volume to 1000 mL, and store at 4°C.

[0112] (2) Fix and permeabilize the cells. The experimental steps refer to the fixation and permeabilization procedures in immunoconfocal experiments.

[0113] (3) Blocking: Vortex the blocking solution, add 1 drop (about 40 μl) of blocking solution to each slide, and incubate it in a constant temperature incubator at 37°C for 60 min.

[0114] (4) Primary antibody incubation: The experimental steps refer to the immunoconfocal experiment.

[0115] (5) Incubate Duolink in situ PLA probes: Vortex PLUS and MINUS PLA probes and PLA probe diluent, dilute PLUS and MINUS PLA probes in PLA probe diluent at a ratio of 1:5. For a 40 μL system, take 8 μL PLUS probe stock solution, 8 μL MINUS probe stock solution and 24 μL probe diluent, and vortex to mix. Discard the primary antibody solution on the slide, wash the slide twice with buffer A, 5 minutes each time. Add 40 μL PLA probe solution to each slide and incubate at 37°C for 120 minutes.

[0116] (6) Ligation: Mix 5× ligation buffer, dilute 5× ligation buffer with high-purity water at a ratio of 1:5 and mix. Pipette the probe solution on the slide and discard it. Wash the slide twice with buffer A for 5 minutes each time. During the washing of the slide, remove the ligase and add the ligase to 1× ligation buffer at a ratio of 1:40. Mix thoroughly, add 40 μL of ligase solution to each slide, and incubate at 37°C for 30 minutes.

[0117] (7) Amplification: Mix 5× amplification buffer, dilute 5× amplification buffer with high-purity water at a ratio of 1:5 and mix. Pipette out the ligase solution and discard it. Wash the slide twice with buffer A for 5 minutes each time. During the washing of the slide, remove the polymerase and add the polymerase to 1× amplification buffer at a ratio of 1:80. Mix thoroughly, add 40 μL of polymerase solution to each slide, and incubate at 37°C for 100 minutes.

[0118] (8) Washing: Dilute part of the 1× wash buffer B to 0.01×. Pipette out the amplification solution on the slide and discard it. Wash the slide twice with 1× wash buffer B, each wash for 5 min, and then wash twice with 0.01× wash buffer B, each wash for 1 min.

[0119] (9) The DAPI staining and sealing steps are the same as those of the immunoconfocal experiment, followed by fluorescence imaging analysis.

[0120] 2.8 Co-immunoprecipitation

[0121] (1) Prepare IP Buffer (4 mL): 2× lysis buffer 2000 μL, double distilled water 1360 μL, 10× PI 400 μL, NaF 80 μL, Na3VO4 80 μL, ppi 80 μL.

[0122] The formula of 2×lysis Buffer is as follows: 2% NP-40, 20% glycerol, 0.27 M NaCl, 1 M Tris at pH 8.0, and the rest is made up with double distilled water.

[0123] (2) Discard the original cell culture medium of the pretreated cells and place them on ice. Add 500 μL of 1× lysis buffer to each well, mix well, and lyse on ice for about 20 minutes. After lysis, blow off the cell fragments at the bottom of the cell culture plate, collect them in a precooled EP tube, and centrifuge them at 4°C and 12,000 rpm for 20 minutes.

[0124] (3) Prepare two batches of EP tubes, one for the IP group and the other for the Input group. The IP group consists of 1×lysis buffer (50μL), 25% BSA (5μL) and antibody (0.3μL). The Input group consists of 6×Loading buffer (8μL). After centrifugation, pipette 40μL of supernatant and add it to the corresponding EP tube of the Input group. After oscillation and mixing, place it in a metal bath and boil it at 99℃ for 10min, and store it at -20℃. Transfer all the remaining supernatant to the EP tube of the IP group, place it on a rotary shaker and shake for 60min, then add 25μL of Protein A / G beads to each EP tube, place it on a shaker at 4℃ overnight or shake it at room temperature for 3h.

[0125] (3) On the second day, balance the IP group and centrifuge at 4°C, 3000 rpm for 1 min. Then dilute the 2× washing solution to 1× with ddH2O. After centrifugation, carefully discard the supernatant. Add 500 μL of 1× washing solution to each EP tube, shake and wash for 1 min, centrifuge at 4°C, 3000 rpm for 1 min, and repeat the above washing method 3 times. Drain the supernatant, draw 20 μL of 1× Loading buffer and add it, mix well, boil in a metal bath at 99°C for 10 min, and then perform the immunoblotting experiment or store in a -20°C refrigerator.

[0126] 2.9 Plasmid construction

[0127] 2.9.1. Design primers based on plasmid sequences

[0128] 2.9.2 PCR

[0129] PCR system: 2×prime starpremix Max 25μL, ddH2O 23μL, Primer antisense 1μL, Primer sense 1μL, Template plasmid 0.3μL.

[0130] The PCR program was set as follows: preheating at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 60°C for 45 s, holding at 72°C for 1 min, 32 cycles, and extension at 72°C for 10 min.

[0131] Use 0.5g agarose + 50μL TAE + 5μL superred dye to prepare 1% agarose gel for use. After the program is completed, add 5μL of 10× Loading buffer to each sample, then load the sample, set a constant voltage of 100V in the horizontal electrophoresis tank, and run for 30 minutes. Cut the required gel according to the size of the target band in the gel imaging instrument and perform gel recovery experiments.

[0132] 2.9.3. Glue recycling

[0133] Use Tiangen's gel recovery kit and follow the instructions.

[0134] 2.9.4. Double enzyme digestion

[0135] Double enzyme digestion system (cutting vector): ddH2O 22μL, plasmid template with vector 3μg, 10×cutbuffer 3μL, enzyme.

[0136] Double enzyme digestion system (gel-cutting recovery product): 10×cutbuffer 3μL, enzyme, gel-recovered DNA product 25μL.

[0137] According to the optimal temperature and time of enzyme cutting of various endonucleases, enzyme cutting is carried out from low temperature to high temperature.

[0138] 2.9.5 Purification

[0139] Use the DNA product purification kit of Tiangen Company and follow the instruction manual.

[0140] 2.9.6 Connection

[0141] Ligation system (10 μL): 7 μL of purified DNA product, 1 μL of 10×Ligase buffer, 1 μL of T4 DNAligase, and 1 μL of vector.

[0142] Ligate for 4 to 6 hours at room temperature or overnight at 4°C.

[0143] 2.9.7 Conversion

[0144] Add 10 μL of the ligation product to 100 μL of competent cells DH5α, mix well, and place on ice for 25 minutes. Place in a 42°C constant temperature water bath, water bath for 90 seconds, and then quickly place on ice for 2 minutes. After the ice bath, pipette 500 μL of LB culture solution into the EP tube, mix well, place in a 37°C constant temperature shaker set at 180 rpm, shake for 45-50 minutes, and centrifuge at 5000 rpm for 2 minutes. Pipette the supernatant, use part of the supernatant to blow and mix the precipitate, and then inoculate the mixture into an LB agar plate containing the corresponding antibiotics and culture it upside down at 37°C.

[0145] 2.9.8. Picking a single clone

[0146] Prepare 5 15mL centrifuge tubes, add 5mL LB culture medium to each tube, and add the corresponding antibiotics in proportion, mix well and set aside. Find a single growing colony on the agar plate, pick it out and add it to a 15mL centrifuge tube, and culture it in a shaking incubator at 37℃ until the LB culture medium turns from clear to turbid.

[0147] 2.9.9 PCR Verification

[0148] Bacterial liquid PCR system: Taq enzyme 10μL, double distilled water 7μL, Primer sense 1μL, Primer antisense 1μL, bacterial liquid 1μL, total volume 20μL.

[0149] Prepare 1% agarose gel, add sample and perform electrophoresis to verify. Determine the result according to the size of the target band.

[0150] 2.9.10 Plasmid extraction

[0151] Use the plasmid extraction kit from Tiangen Company and follow the instructions. After extraction, measure and record the concentration and purity of the plasmid.

[0152] 2.10 Interfering RNA

[0153] Sirt4 siRNA was purchased from GenePharma, and the sequences of the small interfering RNA (siRNA) S1 to S3 used were:

[0154] The sense strand sequence of S1 is 5'-GGGUUAUUUGUGCCAGCAA-3', SEQ ID NO.5; the antisense strand sequence is 5'-UUGCUGGCACAAAUAACCC-3', SEQ ID NO.6;

[0155] The sense strand sequence of S2 is 5'-CAUCCAGCAUGGUGAUUUU-3', SEQ ID NO.7; the antisense strand sequence is 5'-AAAAUCACCAUGCUGGAUG-3', SEQ ID NO.8;

[0156] The forward strand sequence of S3 is 5'-GCGUGUCUGAAACUGAAUU-3', SEQ ID NO.9; the antisense strand sequence is 5'-AAUUCAGUUUCAGACACGC-3', SEQ ID NO.10.

[0157] siRNAPMA-THP1 and HeLa cells were transfected using Lipofectamine 2000 according to the Thermo manufacturer's instructions. 24 hours after transfection, cells were used for further experiments.

[0158] 2.11. Preparation of Sirt4-deficient THP1 cell line

[0159] Sirt4-deficient THP1 cell line was generated by CRISPR / Cas9 technology. Oligonucleotides specifically targeting the Sirt4 gene were used.

[0160] Oligonucleotide 1: 5′-GAAGCCTCATGTGAGAAACTGGG-3′, SEQ ID NO. 1;

[0161] Oligonucleotide 2: 5′-CCACCCCAGTTTCTCACATGAGG-3′, SEQ ID NO. 2;

[0162] Oligonucleotide 3: 5′-AGCTCCACGGATGCATGGACAGG-3′, SEQ ID NO. 3;

[0163] Oligonucleotide 4: 5′-ACTCTCCCAAAACAACACACGGG-3′, SEQ ID NO.4.

[0164] 2.12 Experimental data processing and analysis

[0165] The data of this experiment represent three independent experiments and are expressed as mean ± SD. The present invention performed statistical analysis on different experimental results by using rank sum test and t test. p < 0.05 was considered statistically significant.

[0166] 3. Experimental results

[0167] 3.1. Interaction between Sirt4 and cGAS

[0168] In order to study whether there are members of the Sirtuin family that affect the acetylation modification of cGAS, the present invention used nicotinamide (NAM), a broad-spectrum inhibitor of the Sirtuin family, to conduct a screening experiment. First, PMA-THP1 WT cells (PMA-stimulated monocytes, human macrophage-like cells) were treated with NAM, and it was found that NAM could promote the acetylation modification of cGAS ( Figure 1 A), indicating that the Sirtuin family may be involved in the acetylation regulation of cGAS.

[0169] In order to determine which member of the Sirtuin family is associated with cGAS and affects the acetylation of cGAS, the seven members of the Sirtuin family, Sirt1-Sirt7, were subjected to immunoprecipitation experiments with cGAS in turn. The results showed that only Sirt4 interacted with cGAS ( Figure 1 B~1C).

[0170] Endogenous Co-IP experiments were performed using PMA-THP1 WT cells, which further demonstrated that Sirt4 binds to cGAS, and found that HSV60 (viral DNA derived from HSV-1) stimulation led to upregulation of Sirt4 expression levels and more Sirt4 co-precipitated with cGAS ( Figure 1 D). Fluorescence confocal experiment ( Figure 1 E) and proximity ligation assay ( Figure 1 F) also showed that Sirt4 co-localized with cGAS in cells after HSV-1 infection.

[0171] Next, the interaction domain between cGAS and Sirt4 was investigated. The results showed that the interaction between the cGAS truncation with only the 161-522 amino acid sequence and Sirt4 was significantly weakened, while the interaction between other cGAS truncation forms and Sirt4 was very strong. These data indicate that the interaction domain between cGAS and Sirt4 is mainly located in the 1-160 sequence ( Figure 1 G).

[0172] In order to determine whether the interaction between Sirt4 and cGAS depends on its enzyme activity or is related to its mitochondrial localization, the present invention used Sirt4 enzyme activity-deficient mutant Sirt4 H161Y and mitochondrial localization mutant Sirt4Δ28 to co-transfect HEK293T cells with cGAS plasmids. The results showed that both mutants did not bind to cGAS ( Figure 1 H), indicating that both the enzymatic activity of Sirt4 and its mitochondrial localization are critical for its interaction with cGAS.

[0173] 3.2. Sirt4 knockdown attenuates DNA virus-induced innate immune responses in THP1 cells

[0174] In order to explore the potential function of Sirt4 in DNA virus infection, the present invention first determined whether the expression of Sirt4 was related to DNA virus infection or viral DNA stimulation. PMA-THP1 WT cells were infected with DNA virus HSV-1 or stimulated with viral DNA HSV60, and the expression of Sirt4 was evaluated by immunoblotting. The results showed that both HSV-1 infection and HSV60 stimulation could induce the expression of Sirt4 protein ( Figure 2 A), which suggests that Sirt4 may play a role in the invasion of DNA viruses.

[0175] Next, the present invention evaluated the role of Sirt4 in the innate immunity induced by DNA viruses. The present invention designed Sirt4 siRNA, and explored the function of endogenous Sirt4 in antagonizing the immune response of DNA viruses by knocking down Sirt4. The present invention transfected Sirt4 siRNA (S1, S2, S3) into cells, and the results showed that both S2 and S3 could effectively inhibit the expression level of Sirt4 protein, and S2 had a better effect ( Figure 2 B).

[0176] Next, the present invention knocked down the expression of Sirt4 in PMA-THP1 WT cells, and found through viral titer determination that knocking down Sirt4 promoted the replication of HSV-1 ( Figure 2C). Real-time PCR experiments also showed that knockdown of Sirt4 downregulated the mRNA levels of IFN-β, IP-10, and ISG56 induced by HSV-1 ( Figure 2 D). Knockdown of Sirt4 also inhibited the production of IFN-β and TNF-ɑ when PMA-THP1 WT cells were stimulated with HSV60 ( Figure 2 E). In immunoblotting experiments, knockdown of Sirt4 significantly inhibited the phosphorylation levels of TBK1, IRF3, and p65 in the HSV-1-triggered signaling pathway as well as the aggregation of IRF3 ( Figure 2 F). Through nuclear-cytoplasmic separation experiments, the present invention also found that infection with HSV-1 can significantly promote the nuclear entry of IRF3, while knocking down the expression of Sirt4 significantly inhibited the nuclear entry of IRF3 ( Figure 2 G).

[0177] 3.3 Sirt4 deficiency attenuates DNA virus-triggered innate immune responses in BMDM cells

[0178] By constructing a Sirt4 KO mouse model and isolating and culturing primary bone marrow macrophages, we explored whether Sirt4 also has the function of regulating anti-DNA virus immune response in primary cells. Wild-type and Sirt4-deficient BMDM cells were infected with HSV-1, and the results of enzyme-linked immunosorbent assay showed that Sirt4 knockout significantly inhibited the production of interferon in cells ( Figure 3 A). In addition, Real-time PCR results also showed that Sirt4 knockout also significantly inhibited the mRNA levels of IFN-β, IP-10, ISG56, and TNF-ɑ ( Figure 3 B).

[0179] Western Bolt technology was used to detect the effect of Sirt4 deficiency on the phosphorylation level of related signaling molecules in the DNA virus signal axis. It was found that whether infected with HSV-1 ( Figure 3 C) or HSV60 stimulation ( Figure 3 D) In ​​BMDM cells, the loss of Sirt4 led to a decrease in the phosphorylation levels of TBK1, IRF3, and p65 in the cells.

[0180] 3.4 Sirt4 deficiency impairs DNA virus-triggered innate immune responses in mouse fibroblasts

[0181] The present invention prepared primary mouse embryonic fibroblasts to explore the role of Sirt4 in the innate immune response of non-immune cells infected with HSV-1. Wild-type and Sirt4-deficient MEF cells were infected with HSV-1, and the real-time PCR results showed that Sirt4 deficiency significantly reduced the mRNA levels of IFN-β, TNF-ɑ, and IP-10 in the cells ( Figure 4 A). Western Bolt results showed that the phosphorylation levels of TBK1, IRF3, and p65 induced by HSV-1 were significantly reduced in Sirt4-deficient MEFs compared with wild-type MEF cells ( Figure 4 B).

[0182] Next, the present invention infected MEF cells with HSV-1 and VSV respectively, and through ELISA analysis, it was found that the loss of Sirt4 led to a decrease in the production of IFN-β and IL-6 proteins induced by HSV-1 in the cells, but did not affect the levels of IFN-β and IL-6 proteins induced by VSV in the cells ( Figure 4 C).

[0183] These results suggest that Sirt4 deficiency limits the innate immune response to DNA virus infection in MEFs.

[0184] 3.5 Sirt4 protects mice against HSV-1 infection

[0185] In order to verify the function of Sirt4 in regulating host resistance to DNA viruses, wild-type control mice injected intravenously with HSV-1 and Sirt4 systemic knockout mice were used as infection models to observe the survival rate and weight changes of the two. Compared with wild-type control mice, Sirt4 knockout mice had a higher weight loss rate and lower survival rate after HSV-1 infection ( Figure 5 A and B). ELISA results showed that 6 hours after intravenous injection of HSV-1, the levels of interferon and inflammatory cytokines in the serum of Sirt4 knockout mice were significantly reduced compared with those of wild-type control mice ( Figure 5 C).

[0186] Next, the present invention infected wild-type and Sirt4 knockout mice intranasally with HSV-1 for 24 hours, and then detected the levels of cytokines in bronchoalveolar lavage fluid (BALF). Figure 5 As shown in D, Sirt4 deficiency resulted in decreased secretion of IFN-β, TNF-ɑ, and IL-6 in the bronchoalveolar lavage fluid of mice after infection with HSV-1.

[0187] Finally, the present invention detected the immune response after HSV-1 infection in various organs of wild-type and Sirt4 knockout mice. The results showed that compared with wild-type control mice, the mRNA levels of interferon and inflammatory cytokines in the brain, liver, spleen and lung of Sirt4 knockout mice were decreased, while the HSV-1 gene replication level was increased (HSV-1gDNA)( Figure 6 A~C). The ELISA results are also consistent with this ( Figure 6 B), and the pathological damage caused by HSV-1 infection in the lungs of Sirt4 knockout mice was more severe ( Figure 6 D).

[0188] The above results indicate that Sirt4 protects mice from DNA virus infection by regulating the innate immune response.

[0189] Studies have shown that Sirt4 has some specific deacetylase activities. The mitochondrial trifunctional protein α subunit (MTPα) in hepatocytes is another deacetylated substrate of Sirt4, and its deacetylation by Sirt4 inhibits fatty acid oxidation in the liver. In addition to deacetylase activity, Sirt4 also has ADP-ribosyltransferase activity, lipoamidase and deacylase activity. In different tissues and cells, the substrate and enzyme activity of Sirt4 are not the same, which also leads to its multiple biological functions. The present invention has made new discoveries about the function of Sirt4 in antiviral innate immune response. The present invention found that Sirt4 is a positive regulator of signal pathways triggered by DNA viruses or exogenous DNA. By using HSV-1 to establish mouse and cell infection models, the present invention observed the following findings: First, Sirt4 expression can be induced by HSV-1 infection or viral DNA stimulation. Secondly, at the cellular level, the knockdown or knockout of Sirt4 in immune cells and non-immune cells obtained consistent results, indicating that Sirt4 limits HSV-1 infection and promotes antiviral innate immune response. Finally, in a mouse model infected with HSV-1, the lack of Sirt4 led to a decrease in mouse body weight, survival rate, and antiviral immune responses in various organs. Based on these findings, the present invention concludes that Sirt4 positively regulates the innate immune signaling pathway triggered by DNA viruses or exogenous DNA.

[0190] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of a deacetylase Sirt4 as a drug target for screening drugs for preventing, alleviating or treating HSV-1 viral infection, wherein the application is used for non-disease diagnosis or treatment.

2. A method for screening drugs for preventing, alleviating or treating HSV-1 virus infection, characterized in that: The following steps are involved: The candidate drug is administered to an HSV-1 virus infection model, and the expression level of the deacetylase Sirt4 described in claim 1 is detected before and after administration. The expression level of the deacetylase Sirt4 after administration is lower than the expression level of the deacetylase Sirt4 before administration, which is an indication that the candidate drug is a target drug.

3. A drug for regulating HSV-1 virus infection obtained by screening according to the method of claim 2, characterized in that the drug comprises an expression inhibitor of deacetylase Sirt4, and the expression inhibitor of deacetylase Sirt4 is siRNAS2 or siRNA S3, The sense strand sequence of the siRNA S2 is 5'-CAUCCAGCAUGGUGAUUUU-3', and the antisense strand sequence is 5'-AAAAUCACCAUGCUGGAUG-3'; The sense strand sequence of the siRNA S3 is 5'-GCGUGUCUGAAACUGAAUU-3', and the antisense strand sequence is 5'-AAUUCAGUUUCAGACACGC-3'.

4. The drug according to claim 3, characterized in that The drug is used to improve multiple organ dysfunction syndrome caused by excessive activation of antiviral innate immune response.

5. The drug according to claim 3, characterized in that The drug is used to improve autoimmune diseases caused by excessive activation of antiviral innate immune response.

6. The drug according to claim 3, characterized in that The drug includes pharmaceutically acceptable excipients or carriers.

7. The drug according to claim 6, characterized in that The drug is prepared as an oral preparation or an injection preparation.

8. The drug according to claim 7, characterized in that The oral preparation is a granule, capsule, tablet or liquid preparation.

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