Application of o8G in blocking EV71 virus replication

Abstract

The invention provides application of o8G in blocking EV71 virus replication, and belongs to the technical field of biology. The o8G can well influence the replication of the EV71 virus, can reduce the stability of virus RNA (Ribonucleic Acid), and can provide a new thought for the development of anti-EV71 virus medicines.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to the use of o8G in blocking EV71 virus replication. Background Technology

[0002] When cells undergo pathophysiological changes, oxidative stress usually occurs, resulting in the production of reactive oxygen species (ROS). These free radicals, due to their high reactivity, modify various biological structures, with RNA being the most easily modified. 8-oxoguanine (o8G) is believed to be formed under oxidative stress by the direct oxidation of RNA or guanine by ROS.

[0003] Enterovirus 71 (EV71), belonging to the Picornaviridae family and the Enterovirus genus, is one of the most common pathogens causing hand-foot-and-mouth disease in infants and young children. It is sometimes accompanied by severe central nervous system complications, including aseptic meningitis, brainstem encephalitis, autonomic nervous system disorders, pulmonary edema, and even death. Currently, the prevention and treatment of viral diseases mainly rely on vaccines and drugs. A relevant vaccine was only launched in 2015, and its preventive efficacy still needs further investigation. Therefore, researching novel anti-EV71 virus drugs with high specificity and low toxicity is of significant practical importance for the treatment and prevention of EV71 virus infection and related diseases. Summary of the Invention

[0004] The main objective of this invention is to provide the application of o8G in blocking EV71 virus replication, which can provide a basis for the development of novel anti-EV71 virus drugs and has good application prospects.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: 8-O-guanine was used in the development of drugs to block EV71 virus replication.

[0006] Preferably, the application involves adding pharmaceutically acceptable excipients and carriers to the 8-oxoguanine to prepare an anti-EV71 virus formulation.

[0007] Preferably, the preparation is one of granules, tablets, pills, capsules, injections, and dispersants.

[0008] There are few reports on the antiviral activity of 8-oxoguanine. The only reports indicate that increased 8-oxoguanine and DNA damage can accelerate aging in Polg mutant mice, while its inhibitory activity against EV71 virus has not been reported to date.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) There are currently no antiviral drugs developed for o8G. This invention provides a new approach for the development of antiviral drugs. (2) RNA virus genomes are prone to mutation, which can cause vaccine failure and off-target effects of antiviral drugs. o8G exists conserved in the viral genome. Using it as a target can avoid the problem of drug off-target effects. From this perspective, o8G has broad-spectrum and high-efficiency application value. (3) Through a large number of biological experiments, this invention has found that EV71 RNA is modified with o8G and that o8G has the function of regulating EV71 replication. Specifically, it can promote the proliferation of EV71 in cells, but at the same time it can inhibit the stability of viral RNA, which can provide new ideas for the development of anti-EV71 virus drugs. Attached Figure Description

[0010] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is an experimental result diagram from Example 1 of the present invention demonstrating the presence of o8G modification in EV71 RNA; The image on the left is... Figure 1 A is a real-time quantitative PCR experiment using RIP (re-infection) on RD cells; the image on the right is... Figure 1 B is a real-time quantitative PCR assay performed on Vero cells after infection with RIP.

[0011] Figure 2 Example 2 of this invention demonstrates that the overall o8G modification level of host cells (RD) increases after EV71 infection; Figure 3 Example 3 of the present invention demonstrates that treatment with oxidative agonists and inhibitors can increase and decrease o8G modification on EV71, respectively, and that the binding of PCBP2 and EV71 is increased and decreased under these two treatments, respectively. The image on the left is... Figure 3 Figure A shows the effect of treating EV71-infected cells with NAC and H2O2 on the o8G modification content on EV71 RNA in Example 3; the figure on the right is... Figure 3 B represents the real-time quantitative PCR of PCBP2 and EV71 binding performed on cells infected with EV71 after treatment with NAC and H2O2 in Example 3, using formaldehyde crosslinking RIP.

[0012] Figure 4 This is a diagram from Example 4 of the present invention demonstrating the effect of NAC and PE on EV71 replication after treatment of EV71-infected RD cells. It shows that oxidative inhibitors reduce EV71 replication, while oxidative agonists increase EV71 replication. Figure 5Example 5 of this invention demonstrates that the stability of EV71 RNA containing o8G is lower than that of EV71 RNA without o8G. The image on the left is... Figure 5 A indicates that in Vero cells, the stability of EV71 3D-mut RNA containing o8G is lower than that without o8G. The figure on the right is... Figure 5 B indicates that the same stability-reducing effect is also present in RD cells. Detailed Implementation

[0013] The sources of some of the raw materials used in this invention are as follows: The EV71 XF strain originated from the Wuhan Institute of Virology, Chinese Academy of Sciences; RD cells (American Type Culture Collection [ATCC], Manassas, VA, USA; cat. no. CCL-136), Vero cells (ATCC; cat. no. CCL-81). Anti-o8G (15A3) mouse mAb (4354-MC-050, R&D systems); Phenylephrine (P1240000, Sigma); N-acetylcysteine ​​(A9165-5G, Sigma); 8oxoGTP (N-1066, Trilink); Trizol Reagent (15596018CN, Invitrogen); MEGAscriptT7 Kit (AM1333, Invitrogen); HindIII enzyme was purchased from Themo Scientific; 10× enzyme digestion buffer was purchased from Themo Scientific; Tris saturated phenol was purchased from Qingke Biotechnology.

[0014] The RIP buffer used in this embodiment of the invention is prepared from 150mM NaCl, 10mM Tris-HCl (pH=7.4), 0.1% NP40, 100U / ml RNase inhibitor, 100µM PMSF, and 1µg / ml proteinase inhibitors; IP buffer is prepared from 150mM NaCl, 10mM Tris-HCl (pH=7.4), and 0.1% NP40.

[0015] The formaldehyde crosslinked RIP buffer used in this embodiment of the invention was prepared from 150mM KCl, 25mM Tris-HCl (pH=7.4), 5mM EDTA, 0.5mM DTT, 0.5% NP40, 100U / ml RNase inhibitor, 100μM PMSF, and 1μg / ml proteinase inhibitors.

[0016] The formaldehyde crosslinked RIP washing buffer used in this embodiment of the invention was prepared from 300mM KCl, 25mM Tris-HCl (pH=7.4), 5mM EDTA, 0.5mM DTT, 0.5% NP40, 100U / ml RNase inhibitor, 100μM PMSF, and 1μg / ml proteinase inhibitors.

[0017] Example 1 Experiments demonstrating the presence of o8G modification in EV71 RNA: EV71 virus was inoculated into RD and Vero cells at a moi=0.1. Two hours post-infection, the DMEM medium was removed, and the cells were washed once with PBS. Fresh serum at a concentration of 2% (v / v) was then added, and the cells were incubated at 37°C. Twenty-four hours post-infection, the medium was removed, and the cells were washed once with PBS. After completely aspirating the PBS, Trizol was added at a rate of 3 mL per 10 cm dish. The mixture was pipetted until complete lysis, and all liquid was collected for RNA extraction. The RNA extraction method is as follows: 1. Add 200 μL of chloroform to each 1.5 mL EP tube containing 1 mL of Trizol solution, tighten the cap, shake vigorously up and down for 15 seconds, let stand for 3 minutes, and then centrifuge at 4℃ for 12000 rpm for 15 minutes; 2. After centrifugation, you will see the layers. Carefully remove the centrifuged EP tubes, and take another clean 1.5 mL EP tube without RNase. Use a pipette to carefully pipette 180 µL of the separation supernatant into the new EP tube each time, and pipette twice. Add 500 mL of isopropanol to each EP tube, tighten the cap, invert and mix well, and then centrifuge at 4℃ for 12000 rpm for 15 minutes; 3. Remove the centrifuged EP tubes. You will see a white RNA precipitate at the bottom of the tube. Pour out the solution, avoiding pouring out the white precipitate. Add 1 mL of 75% ethanol (prepared with anhydrous ethanol and DEPC-treated water) to each tube. Invert the tube to wash away the precipitate, then centrifuge at 12000 rpm for 5 minutes at 4°C. Repeat twice. 4. Centrifuge again at high speed to remove any residual liquid from the tube walls. After removing all remaining liquid from the tube, allow it to air dry until the precipitate is translucent. Add 30-100 μL of nuclease-free water (depending on the amount of RNA added; this can be roughly estimated from the size of the white precipitate). Let it stand for 15 minutes, then measure the RNA concentration to ensure consistent subsequent dosages. After measuring the RNA concentration, divide the tubes into two groups: IgG (immunoglobulin G, used as a control for non-specific antibody binding) and o8G. Each group uses 300 μg of Total RNA added to 300 µL of the solution. In RIP buffer, 10 μL of each group was taken as input (control group, containing unbound RNA to correct for the corresponding treatment group). The remainder was incubated overnight at 4°C with Anti-IgG and Anti-o8G, respectively. Then, magnetic beads washed three times with IP buffer were added, and the mixture was incubated at 4°C for 2 hours. After washing six times with IP buffer, 1 mL of Trizol was added, and RNA was extracted again using the method described above.The extracted RNA was reverse transcribed using ABScriptIII RT MasterMix for qPCR with gDNA Remover to obtain cDNA, which was then amplified by qPCR using SYBRGreen Master Mix No Rox.

[0018] The primers used for RT-PCR sequences are shown in the table below:

[0019] Experimental results are as follows Figure 1 As shown, this demonstrates that EV71 RNA is modified with o8G.

[0020] Example 2 This demonstrates that the overall o8G modification level in host RD cells increases after EV71 infection: RD and Vero cells were cultured and divided into two groups: an uninfected (Mock) group and an infected group. The infected group was infected with EV71 at a moi=1. RNA was collected from both the Mock and infected groups after 24 hours. The collection and extraction methods are detailed in Example 1. Equal amounts (10, 20, and 30 μg) of total RNA from uninfected (Mock) and EV71-infected cells were taken and divided into two groups. The RNA was reprecipitated and then dissolved and concentrated in 8 μL of Nuclease-Free water. The reprecipitation method was as follows: add twice the volume of anhydrous ethanol and 1 / 10 volume of 3M NaAC solution to the RNA, incubate overnight at -80°C, and the next day centrifuge at 12000 rpm for 15 minutes at 4°C until a white precipitate is observed. Remove the solution outside the precipitate and allow it to air dry until the precipitate is translucent. Then, add 8 μL of Nuclease-Free water to dissolve the RNA. The dissolved RNA was denatured in a 75°C metal bath for 5 minutes and then quickly placed on ice for 1 minute. Then it was dropped onto a Hybond-N+ membrane, allowed to air dry naturally, and then subjected to three UV crosslinking processes (150 MJ / cm). 2 After (254 nm), the membrane was placed in a 5% milk blocking buffer prepared with 0.1% PBST (PBS containing 0.1% Tween-20). After incubation on a shaker at room temperature for 1 hour, antibody was added to the 5% milk at a concentration of 1:250 as the primary antibody incubation solution. The primary antibody incubation solution and the cross-linked Hybond-N+ membrane were incubated overnight at 4°C. The membrane was washed three times with 0.1% PBST. The HRP secondary antibody was diluted 1:5000 with blocking buffer according to the antibody instructions. The membrane was incubated with the washed membrane at room temperature for 1 hour, followed by three more washes with 0.1% PBST. Color development was then performed, and the test results are shown below. Figure 2 As shown: This illustrates the enhanced o8G modification in EV71 infection.

[0021] Example 3 The study demonstrated that treatments with oxidative agonists and inhibitors could increase and decrease o8G modification on EV71, respectively, and that the binding of PCBP2 and EV71 was increased and decreased under these two treatments, respectively. In this embodiment, the oxidative agonist H2O2 and the inhibitor N-acetylcysteine ​​(NAC) were used to oxidatively activate and inhibit the entire host system infected with the virus, respectively, to increase and decrease the o8G content in the EV71 genome in the host. Vero cells were used and divided into three groups: a control group (NT group), an H2O2 group, and an NAC (N-acetylcysteine) group. When the cell density reached ≥80%, all cells were infected with EV71 moi=1. Six hours after infection, the cells were treated accordingly: The H2O2 group was treated with 100 mM H2O2 at 37°C for 5 minutes, then completely washed with PBS, and replenished with 2% DMEM medium (8 mL per 10 cm dish); the NAC group had its original medium removed, was washed with PBS, and then diluted with 2% DMEM medium to a final concentration of 2 mM (8 mL per 10 cm dish), without washing; the NT group had its original medium removed, was washed with PBS, and then replenished with 2% DMEM medium (8 mL per 10 cm dish). After treatment, the cells were cultured for another 24 hours. The medium from all three treatments was completely washed away with PBS, and Trizol (3 mL per 10 cm dish) was added for RNA extraction, following the procedures described in Example 1. The RIP method from Example 1 was used for testing (from the addition of total RNA to RIP buffer until qPCR completion), and the results are as follows. Figure 3 As shown in A; Two susceptible cell types, RD and Vero cells, were treated with oxidative agonists and inhibitors, respectively, to increase and decrease the o8G content in the virus. The treatment methods were the same as described in this example, and three groups (NT, NAC, and H2O2) were set up for infection. 24 hours post-infection, cross-linking was performed using 1% formaldehyde, 5 mL per 10 cm dish. The mixture was treated at 37°C for 10 min, and then 2.5 M glycine solution was added to achieve a final concentration of 0.125 M, i.e., 250 µL, added to the preceding 1% formaldehyde solution. After incubating at 37°C for 5 min, wash three times with PBS to remove residual liquid. Add 1 mL of pre-prepared formaldehyde crosslinking RIP buffer per dish on ice. Scrape cells using a cell scraper and collect them in 1.5 mL EP tubes. Incubate on ice for 30 min, then centrifuge at 1600 rpm for 10 min at 4°C. Take 100 mL of supernatant as input. Transfer the remaining supernatant to new EP tubes as the treatment group. Add 4 μL of PCBP2 protein antibody to each tube and incubate overnight at 4°C to achieve antibody-protein-RNA binding. Then, wash the Prtein G Agorose Beads three times with the prepared RIP buffer and incubate at 4°C for 2 h to fully bind to the RNA-protein complex. Wash the Beads three times with formaldehyde crosslinking RIP buffer, and finally wash three times with RIP washing buffer. Aspirate the supernatant, add 1 mL of Trizol per 1.5 mL EP tube, and extract RNA to obtain protein-bound RNA. The relative quantification analysis of RT-qPCR described in Example 1 was performed, and the experimental results are as follows: Figure 3 As shown in Figure B, the results indicate that PCBP2 exhibits a consistent trend with oxidative activating and inhibiting processes, suggesting that PCBP2 is an o8G reading protein.

[0022] Example 4 Demonstrating the effects of NAC (N-acetylcysteine) and PE (phenylephrine) treatment on EV71-infected RD cells on EV71 replication: Following the method described in Example 3, RD cells were treated and infected using oxidative activating agents (phenylephrine, PE) and inhibitory agents (N-acetylcysteine, NAC). RNA was then extracted and detected using the same reverse transcription and qPCR methods. The results are as follows: Figure 4 As shown, compared with the NT group (control group without oxidative agonist or inhibitor treatment), EV71 replication was enhanced after RD cell infection with PE treatment; EV71 replication was reduced after EV71-infected RD cells were treated with NAC. These results indicate that increased o8G of EV71 RNA promotes EV71 replication, while decreased o8G inhibits EV71 replication.

[0023] Example 5 The results show that the stability of EV71 RNA containing o8G is lower than that of EV71 RNA without o8G: Infectious clones of EV71 3D mutants (plasmids capable of transcribing EV71 RNA with 3D deletion of the key EV71 replication gene) preserved in the laboratory were used to transcribe EV71 3D-mut RNA incorporating 100% GTP, 80% o8GTP, and 20% GTP in vitro using the MEGAscriptT7 Kit, respectively, as the GTP and o8GTP groups. The specific steps for in vitro transcription are as follows: 1. Take 10 µg of an infectious clone of EV71 3D mutation, add 1 µL of HindIII enzyme and 1 µL of 10× digestion buffer, and add ddH2O to a total volume of 10 µL. Digest overnight in a 37°C water bath; 2. Add 500 µL of Nuclease-Free water to the sample from step 1, add 500 µL of Tris-saturated phenol, shake vigorously to mix, and centrifuge at 12000 rpm for 5 minutes at 4°C. Clear separation will be observed. Carefully aspirate all the supernatant liquid into a new EP tube; 3. Add 500 µL of chloroform, shake vigorously to mix, and centrifuge at 12000 rpm for 5 minutes at 4°C. Clear separation will be observed. Carefully aspirate up to 400 µL of the supernatant liquid into a new EP tube, add twice the volume of the aspirated liquid in anhydrous ethanol and 1 / 10 volume of 3M ethanol. NaAC solution was incubated overnight at -80℃. The next day, it was centrifuged at 12,000 rpm for 15 minutes at 4℃ until a white precipitate was observed. The solution outside the precipitate was removed, and the mixture was allowed to air dry until the precipitate was translucent. 17 μL of Nuclease-Free water was added to dissolve the precipitate, and the concentration was measured. 4. 1 µg of the template dissolved in step 3 was taken, and 1 µL each of ATP, CTP, UTP, and GTP were added (for the o8GTP group, 0.8 µL of o8GTP and 0.2 µL of GTP were added, and the rest were the same), as well as 1 µL of 10×Reaction Buffer and Enzyme Mix. The system was brought to 10 µL with Nuclease-Free water, and the reaction was carried out in a 37℃ water bath for 6 hours. 5. 1 µL of TURBO DNase was added, and the reaction was carried out in a 37℃ water bath for 15 minutes. Add 30µL LiCl Precipitation Solution and 2µL glycogen, mix well, and incubate overnight at -80℃; 6. The next day, centrifuge at 4℃, 12000rpm for 15 minutes, remove the supernatant, wash once with the 75% ethanol mentioned above, centrifuge at 12000rpm for 5 minutes, aspirate the supernatant, air dry the ethanol, add 50µL Nuclease-Free water to dissolve for 15 minutes, and then measure the RNA concentration for subsequent experiments.The culture medium from the confluent cell plates was poured out, and the plates were washed three times with Opti-MEM. 250 μL of Opti-MEM, 1 μg of in vitro transcribed RNA, and 4 μL of DMRIE-C transfection reagent were mixed in each well and added to the corresponding wells of RD and Vero cell plates. The plates were incubated at 37°C. After 4 hours of incubation, the liquid in the wells was poured out, and the plates were washed with complete culture medium. RNA was collected at 0h, 2h, 4h, and 6h after 30 minutes. Reverse transcription was performed, and relative quantification was performed using qPCR, following the same procedure as in Example 1. The linear fit of the results is shown below. Figure 5 As shown, Figure 5 A indicates that in Vero cells, the stability of EV71 3D-mut RNA containing o8G is lower than that without o8G. Figure 5 B indicates that the same effect is observed in RD cells, suggesting that the stability of EV71 RNA incorporating o8GTP is significantly reduced.

[0024] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. 8-O-guanine, characterized in that: It will be applied to the development of drugs that block EV71 virus replication.

2. The application according to claim 1, characterized in that: Pharmaceutically acceptable excipients and carriers are added to the 8-oxoguanine to prepare an anti-EV71 virus formulation.

3. The application according to claim 2, characterized in that: The preparation is one of the following: granules, tablets, pills, capsules, injections, and dispersants.

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