Application of N4-acetylcytidine in prevention and treatment of RNA (Ribonucleic Acid) virus

Abstract

The invention discloses application of N4-acetylcytidine in prevention and treatment of RNA (Ribonucleic Acid) viruses, belongs to the technical field of biological medicines, and particularly discloses application of N4-acetylcytidine in prevention and treatment of RNA viruses, and the N4-acetylcytidine is used for blocking RNA polymerase replication on which virus RNA depends. The RNA virus genome is easy to mutate, so that the vaccine failure and the off-target of the antiviral drug can be caused, but the action mode of the virus RdRp is extremely conservative, so that the replication process of the virus is inhibited from the RdRp, a new thought is provided for the development of specific and efficient anti-RNA virus drugs, and the application of N4-acetylcytidine is also widened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of N4-acetylcytidine in prevention and treatment of RNA viruses. BACKGROUND

[0002] Chemical modification is conserved in the RNA of all living things, has an important regulatory effect on the metabolism and function of RNA, and is a hotspot in the field of epigenetic research in recent years.

[0003] N4-acetylcytidine (Chinese name: N-acetylcytosine riboside, abbreviation: ac4C) is the only known acetylation modification in eukaryotic RNA, and early researches on it mainly focused on tRNA and rRNA. Until the discovery of ac4C in mRNA in 2018, this modification has become another frontier hotspot in the field of epigenetic research.

[0004] ac4C widely exists in tRNA, rRNA and mRNAs of eukaryotes and prokaryotes, and plays multiple roles in cell processes. ac4C in tRNA enhances protein translation fidelity and tRNA stability; ac4C in rRNA is extremely important for maintaining protein translation accuracy and rRNA biosynthesis; ac4C in mRNA is relatively high in abundance near the translation initiation codon, which can enhance mRNA stability, but has a complex effect on protein translation: (1) ac4C modification in the protein coding region of mRNA promotes translation, (2) ac4C in the 5' untranslated region (UTR) enhances upstream translation initiation, but ac4C in the Kozak sequence changes the structure of mRNA and tRNAiMet binding, thereby inhibiting the classic translation initiation.

[0005] ac4C was discovered in 1960, but whether this modification exists in viral RNA has not been reported for a long time. Until 2018, researchers found that ac4C may be widely present in viral RNA, suggesting that ac4C has an important function in the process of viral infection, but the function of ac4C in viral infection is less studied.

[0006] At present, there is a lack of specific and efficient drugs for preventing and treating RNA virus infection in clinical practice, and symptomatic treatment is often used. The marketed RNA virus vaccine cannot cope with the rapidly changing pathogen spectrum, and has potential safety factors. So far, various RNA virus infections have caused more serious symptoms, but the pathogenesis is not clear, therefore, it is imperative to develop specific drugs for preventing and treating RNA viruses. SUMMARY

[0007] In view of the above existing problems, the present application provides a new idea for specific and efficient antiviral drug development by using N4-acetylcytidine (ac4C) to block the RNA-dependent RNA polymerase replication process of viruses from the perspective of virus epigenetic modification.

[0008] To achieve the above-mentioned purpose, the present application provides an application of ac4C in preventing and treating RNA viruses, and uses the ac4C to block the RNA-dependent RNA polymerase (RdRp) replication of viruses.

[0009] As a preferred solution, the virus includes enterovirus 71 (EV71).

[0010] Most RNA viruses replicate their genomes by encoding RdRp. Although it has been proved that RNA modification regulates viral replication, there are few studies on whether RNA modification directly affects the rate of RNA synthesis by viral RdRp. Some studies have reported that Nm modification in RNA can reduce the replication rate of flavivirus RdRp (NS5 protein) in in vitro experiments, but the specific mechanism has not been studied.

[0011] The present application studies the function of ac4C in the replication process of viral polymerase (RdRp) by taking enterovirus 71 as an example. EV71 belongs to the Enterovirus genus of the Picornaviridae family, has no envelope, and has a single-stranded positive RNA genome of about 7.4 kb. It includes a structured 5' untranslated region (UTR), a coding region, and a 3' UTR with a poly(A) tail. During the infection process, the RNA genome of EV71 is directly translated into a large polyprotein as mRNA, which is cut into 4 structural proteins (VP1-4) and 7 non-structural proteins (2A-C, 3A-D) by viral proteases 2A and 3C. Among them, the 3D protein is the polymerase (RdRp) of EV71, which replicates the EV71 genome to produce negative-strand RNA, and then uses the negative-strand RNA as a template to produce new progeny virus RNA genome. In vivo, the initiation of RdRp replication usually starts from the tyrosine residue on the VPg (3B) protein, while in vitro, the purified RdRp can bind to the double-stranded structure formed by the template-RNA primer pair and extend from the primer end. In vitro experiments using purified EV71 RdRp and T33-1 template prepared by T7 in vitro transcription found that ac4C located on the primer pairing region or the extension region of the template strand can inhibit the RNA synthesis rate of RdRp, and it was proved that ac4C located in the extension region has a more significant impact on RdRp. When Mn 2+, the inhibition of ac4C on the replication of RdRp was significantly weakened. By reaction band analysis, it was found that the replication reaction was more inclined to stop at the pairing extension of the nucleotide after ac4C. When ac4C TPs were used as substrates to replace CTPs, there was no inhibition on the replication of RdRp, indicating that ac4C located in the primer chain did not affect the replication function of RdRp. These studies show that ac4C has a very obvious effect on the replication process of RdRp, so ac4C has potential application value in the specific inhibition of EV71 replication, and reflects the potential of ac4C as an antiviral drug target in the replication process of RNA virus.

[0012] An RNA virus prevention and treatment drug, characterized in that the drug uses N4-acetylcytidine as a drug target.

[0013] Compared with the prior art, the beneficial effects of the present application are: (1) At present, there is no antiviral drug developed for RNA modification, and the present finding provides a new idea for the development of antiviral drugs from the perspective of viral epigenetic modification.

[0014] (2) The RNA virus genome is prone to mutation, which can cause vaccine failure and off-target of antiviral drugs, however, the mode of action of viral RdRp is extremely conservative, therefore, the present application starts from RdRp to inhibit the replication process of the virus, providing a new idea for the development of specific and efficient antiviral drugs.

[0015] (3) The current functions of ac4C modification include affecting the temperature and translation efficiency of RNA, and there is no report on its function in the specific process of RdRp replication, and the present study found that ac4C affects the specific process of EV71 RdRp replication, expanding the function research of ac4C. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a schematic diagram for T33-1 template preparation and RdRp in vitro replication experiment.

[0017] Figure 2 is a graph of ac4C (+) T33-1 template inhibiting the replication rate of RdRp.

[0018] Figure 3 is a long-time reaction graph of RdRp with ac4C (+) T33-1 template.

[0019] Figure 4 is a schematic diagram for T33-2 template and RdRp in vitro replication experiment.

[0020] Figure 5 is a graph of ac4C (+) T33-2 template inhibiting the replication rate of RdRp.

[0021] Figure 6 Graph comparing the effect of ac4C(+) T33-1 and T33-2 templates on RdRp inhibition.

[0022] Figure 7 Graph comparing the effect of ac4C(±) T33-1 and T33-2 templates on RdRp rate. DETAILED DESCRIPTION

[0023] The technical solutions of the present application are further described and explained below by way of examples. The raw materials used in the examples are commercially available or prepared by conventional methods.

[0024] Example 1 1. Preparation of RNA template S1, produce DNA containing T7 promoter, T33-1 or T33-2 and glmS ribozyme sequence by PCR (Polymerase Chain Reaction); S2, use the PCR product in step S1 as a template to perform a transcription reaction in a 5 mL volume of transcription buffer (60 mM HEPES, pH 7.8, 2 mM spermidine, 0.01% Triton X-100, 15 mM DTT, 22 mM MgCl2, 4 mM each NTP or ac4CTP, and 25 mg / mL T7 RNA polymerase) to perform the reaction; S3, incubate the sample obtained in step S2 at 37°C for 4 hours, and then treat with TURBO DNAse at 37°C for 30 minutes; S4, perform electrophoresis on the sample obtained in step S3 using a 10% polyacrylamide / 7M urea gel, and after the electrophoresis is complete, cut out the gel in the region where the full-length RNA template is located; S5, cut the gel in the region where the full-length RNA template is located in step S4 into pieces and add RNA elution buffer (300 mM sodium acetate, 1 mM EDTA [pH 8.0] and 100 U / mL RNasin), and let stand at 4°C overnight; S6, purify the sample obtained in step S5 using a Spin-X column (Corning, NY, USA), precipitate with isopropanol, and after washing with 75% ethanol, obtain the template, dissolve the obtained template in RNA annealing buffer, and after incubation at 95°C for 3 minutes, store at -80°C.

[0025] 2. In vitro RdRp RNA synthesis assay P1, ac4C (+) template, ac4C (-) template and P10 primer were mixed at a ratio of 1:1.1, incubated at 45 °C for 3 min, then slowly cooled to room temperature to anneal the primer to the template to form a template-primer complex; P2, the template-primer complex prepared in step P1 was added to the reaction mixture containing 50 mM HEPES (pH = 7.0), 50 mM KCl, 5 mM MgCl2(or 4 mM MgCl2and 1 mM MnCl2), 5 mM TCEP, 300 μM each NTP (GTP and ATP for T33-1 reaction, CTP / ac4CTP and UTP for T33-2 reaction) and 6 μM His-RdRp at a final concentration of 4 μM (the concentration of template-primer complex in the final reaction system is 4 μM), and incubated at 22.5 °C for the specified time (reaction time from 0.25 min to 60 h, see Figure 2 , Figure 3 , Figure 5 , Figure 6 , Figure 7 ); P3, when the reaction in step P2 reached the specified time, the same volume of stop solution as the solution in step P2 was added to quench the reaction, and the components of the stop solution included: 95wt% formamide, 20 mM EDTA (pH = 8.0), 0.02wt% bromophenol blue and 0.02wt% xylene cyanol; P4, the mixture prepared in step P3 was subjected to gel electrophoresis using 20% polyacrylamide / 7M urea gel; P5, the product obtained in step P4 was stained using Stains All (Sigma-Aldrich) stain to visualize the RNA bands.

[0026] 3. Results In the in vitro RNA synthesis experiment with RdRp, the purified EV71 RdRp was mixed with the template-P10 primer mixture (see Figure 1 ), when GTP and ATP were provided, RNA synthesis began, and the P10 primer was extended to produce a 14-mer product (P14). The experimental results showed that in the ac4C modified template, almost no P14 product was detected within 30 minutes compared to the unmodified template (see Figure 2 ), which indicated that ac4C modification significantly inhibited RNA synthesis at the initial stage. In addition, as the reaction time increased, the level of P14 product gradually increased (see Figure 3), indicating that although ac4C modification initially reduces RNA synthesis, this inhibition can be attenuated over time. This finding reveals the dynamic effect of ac4C modification on RNA synthesis and can provide new insights into understanding the role of RNA modification in the regulation of gene expression.

[0027] ac4C was present in both the P10 pairing region and the extension region of the T7 transcription template with ac4C-CTP as substrate Figure 1 The present application designed to synthesize T33-2 template for reaction, T33-2 does not contain ac4C modification in the extension region (GAGA, Figure 4 ), to analyze the effect of two ac4C sites in the primer pairing region on the efficiency of RdRp synthesis. Studies have found that the P14 product level of the template ac4C (+) template containing ac4C modification is lower than that of the unmodified template ac4C (-) template Figure 5 ), indicating that the two ac4C sites in the primer pairing region slow down the efficiency of RNA synthesis. The present application also compared the effect of T33-2 and T33-1 containing ac4C modification on the replication rate of RdRp, and found that T33-2 has a faster reaction speed Figure 6 ), indicating that the two ac4C modifications in the extension region play a key role in slowing down RNA synthesis. Compared with the unmodified template, the RNA synthesis of T33-1 template using ac4C modification is reduced by about 89% Figure 7 A), while the RNA synthesis of acetylated T33-2 is reduced by about 38% Figure 7 B), indicating that ac4C in the primer region and the extension region reduces the RNA synthesis of RdRp, and ac4C in the extension region has a more obvious effect.

[0028] In summary, the experimental results show that ac4C modification has different effects on the efficiency of RNA synthesis in different regions, especially the ac4C modification in the extension region has a more significant effect on slowing down RNA synthesis. These findings are of great significance for understanding how RNA modification regulates gene expression.

[0029] It should be understood that the above examples are only used to illustrate the content of the present application and not to limit the protection scope of the present application. In addition, it should be understood that after reading the content taught by the present application, those skilled in the art can make various modifications or modifications to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. Use of N4-acetylcytidine for the prevention and treatment of RNA viruses, characterized in that, The viral RNA-dependent RNA polymerase replication is blocked by the N4-acetylcytidine.

2. Use according to claim 1, characterized in that, The virus includes enterovirus 71.

3. A medicine for preventing and treating RNA virus, characterized by, The drug takes the N4-acetylcytidine as a drug target.

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