Application of methyltransferase 3 as target for treating or inhibiting Ebola virus

Abstract

The invention relates to the field of biological medicine, in particular to application of methyltransferase 3 as a target spot for treating or inhibiting Ebola viruses, and finds that the expression of the methyltransferase 3 gene can be interfered by using siRNA of the methyltransferase 3 gene, and the proliferation of the Ebola viruses can be inhibited. Moreover, by using the methyltransferase 3 gene inhibitor STM2457, the proliferation of the virus, the methylation of the virus, the expression of the virus VP40 protein and the formation of virus inclusion bodies (IBs) can be obviously inhibited, so that the proliferation of the Ebola virus can be inhibited by inhibiting the methyltransferase 3 gene; it is prompted that the methyltransferase 3 gene can be used as a target spot for treating the Ebola virus disease.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to the application of methyltransferase 3 as a target for treating or inhibiting Ebola virus. Background Art

[0002] Ebola virus (EBOV) is a non-segmented single-stranded negative-sense RNA virus belonging to the Filoviridae family. It is a highly contagious virulent virus that seriously threatens human health. EBOV includes 7 structural genes, encoding 7 structural proteins, namely nucleoprotein (NP), polymerase cofactor (VP35), matrix protein (VP40), glycoprotein (GP), transcription activator (VP30), minor matrix protein (VP24) and RNA-dependent RNA polymerase (L). Ebola virus is the pathogen that causes Ebola virus disease (EVD) in humans and non-human primates (NHPs), causing fever, disseminated intravascular coagulation (DIC), lymphocyte apoptosis, abnormal bleeding and multiple organ failure, and can lead to death in severe cases. During the assembly process, viral RNA forms a ribonucleoprotein (RNP) complex with NP, L, VP30 and VP35 to form a helical nucleocapsid (NC) that aggregates in inclusion bodies (VIBs). Inclusion bodies (IBs) are very important in viral proliferation. Viral nucleocapsid formation and RNA replication occur in inclusion bodies, which are important sites for viral replication and proliferation. Fluorescent staining VP35 can indicate the location and size of viral inclusion bodies.

[0003] Methyltransferase-like 3 (METTL3) is the first m6A methyltransferase discovered. Its protein size is about 70 kda and is highly conserved in eukaryotes from yeast to humans. It, together with METTL14 and WTAP, constitutes the m6A methyltransferase complex. The three protein components perform their respective functions, with clear division of labor and close cooperation. METTL14 provides a platform for RNA binding, WTAP is responsible for recruiting the complex to the RNA target, and METTL3 is the core component of the complex's catalytic activity. As the core protein in the m6A methyltransferase complex, METTL3 can catalyze the methylation of the sixth nitrogen atom of adenine (A) on RNA and participate in important biological processes such as RNA assembly, translation, nuclear export, and degradation. Therefore, METTL3 is involved in a variety of physiological processes, including cell proliferation, apoptosis, immunity and inflammation, tumor invasion and differentiation, etc.

[0004] METTL3 has become an important target for the treatment of tumors, especially hematological tumors. STM2457 is a highly efficient and specific inhibitor targeting METTL3, which is clinically used for the treatment of leukemia.

[0005] METTL3 plays an important role in regulating viral proliferation and anti-viral infection. It can promote mRNA stability and protein transcription through m6A methylation modification, promote the expression of immune proteins, and thus participate in the innate immune process. Currently, there are no reports on using methyltransferase 3 as a target for the treatment of Ebola virus. Summary of the invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and provide an application of methyltransferase 3 as a target for treating or inhibiting Ebola virus.

[0007] To achieve the above purpose, the technical solution adopted by this application is: The present application provides an application of a reagent for knocking out the methyltransferase 3 gene or a reagent for inhibiting the expression of the methyltransferase 3 gene in any one of the following a1)-a4): a1) Preparation of products for the treatment or adjunctive treatment of Ebola virus disease; a2) Preparation of products that inhibit Ebola virus infection; a3) Preparation of products that inhibit the proliferation of Ebola virus; a4) Preparation of products for the prevention of Ebola virus disease.

[0008] It has been found that knocking down or knocking out the methyltransferase 3 gene or inhibiting the methyltransferase 3 gene can significantly inhibit the proliferation of Ebola virus in cells. The present application provides a reagent for knocking out the methyltransferase 3 gene or an agent for inhibiting the expression of the methyltransferase 3 gene, which can significantly inhibit the m6A methylation level of Ebola virus and inhibit the proliferation of Ebola virus. It shows that the methyltransferase 3 gene can be used as a candidate target for the development of anti-EBOV drugs, which has important application value for the treatment of Ebola virus disease.

[0009] As a preferred embodiment of the application described in the present application, the reagent for inhibiting the expression of the methyltransferase 3 gene includes a methyltransferase 3 gene inhibitor or a methyltransferase 3 gene siRNA.

[0010] As a preferred embodiment of the application described in the present application, the methyltransferase 3 gene inhibitor includes the STM2457 compound, the structural formula of which is shown in Formula I.

[0011] Formula I.

[0012] As a preferred embodiment of the application described in the present application, the concentration of the STM2457 compound is 1-10 μM.

[0013] The STM2457 compound used in this application can significantly inhibit the m6A methylation level of Ebola virus and inhibit the proliferation of Ebola virus, indicating that the methyltransferase 3 gene inhibitor-STM2457 compound can be used to inhibit the proliferation of Ebola virus or treat Ebola virus disease.

[0014] As a preferred embodiment of the application described in the present application, the methyltransferase 3 gene siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID No: 1.

[0015] The present application uses siRNA of the above nucleotide sequence to interfere with the expression level of the methyltransferase 3 gene in cells and can significantly inhibit the proliferation of Ebola virus.

[0016] As a preferred embodiment of the application described in the present application, the reagent for knocking out the methyltransferase 3 gene is a CRISPR / Cas9 gene editing system for knocking out the methyltransferase 3 gene.

[0017] Regarding whether methyltransferase 3 can be used to inhibit Ebola virus, it has been found that the present application provides methyltransferase 3 as a target for treating or inhibiting Ebola virus and related products.

[0018] The present application found that the use of methyltransferase 3 gene siRNA can interfere with the expression of methyltransferase 3 gene and inhibit the proliferation of Ebola virus.

[0019] The use of the methyltransferase 3 gene inhibitor STM2457 can also significantly inhibit the proliferation of the virus, inhibit the methylation of the virus, inhibit the expression of the viral VP40 protein, and inhibit the formation of viral inclusion bodies (IBs). This shows that the proliferation of the Ebola virus can be inhibited by inhibiting the methyltransferase 3 gene, suggesting that the methyltransferase 3 gene can be used as a target for the treatment of Ebola virus disease, and the methyltransferase 3 gene inhibitor STM2457 can be used to treat or inhibit Ebola virus disease.

[0020] The present application also provides the use of an agent for inhibiting the activity of methyltransferase 3 protein or an agent for reducing the content of methyltransferase 3 protein in any of the following b1)-b4): b1) Preparation of products for the treatment or adjunctive treatment of Ebola virus disease; b2) Preparation of products that inhibit Ebola virus infection; b3) Preparation of products that inhibit the proliferation of Ebola virus; b4) Preparation of products for the prevention of Ebola virus disease.

[0021] As a preferred embodiment of the application described in the present application, the reagent for inhibiting the activity of methyltransferase 3 protein or the reagent for reducing the content of methyltransferase 3 protein is siRNA or methyltransferase 3 gene inhibitor with a nucleotide sequence as shown in SEQ ID No: 1.

[0022] The present application also provides the use of a methyltransferase 3 gene inhibitor in the preparation of a drug for inhibiting the formation of Ebola virus inclusion bodies, wherein the methyltransferase 3 gene inhibitor includes the STM2457 compound.

[0023] The present application also provides the use of a methyltransferase 3 gene inhibitor in the preparation of a reagent for inhibiting the m6A methylation level of Ebola virus, wherein the methyltransferase 3 gene inhibitor includes the STM2457 compound.

[0024] The present application also provides the use of a methyltransferase 3 gene inhibitor in the preparation of a reagent for inhibiting the expression level of the Ebola virus protein VP40, wherein the methyltransferase 3 gene inhibitor includes the STM2457 compound.

[0025] Studies have found that the use of the methyltransferase 3 gene inhibitor STM2457 compound can significantly inhibit the m6A methylation level of Ebola virus and inhibit the proliferation of Ebola virus.

[0026] The use of the methyltransferase 3 gene inhibitor - STM2457 compound can significantly inhibit the expression of Ebola virus protein VP40.

[0027] The use of the methyltransferase 3 gene inhibitor - STM2457 compound can significantly inhibit the formation of Ebola virus inclusion bodies, indicating that the methyltransferase 3 gene can be used as a target to inhibit the proliferation of Ebola virus or treat Ebola virus.

[0028] The present application also provides a biomaterial, which includes an agent for inhibiting the expression of methyltransferase 3 gene, and the agent for inhibiting the expression of methyltransferase 3 gene includes STM2457 compound or methyltransferase 3 gene siRNA with a nucleotide sequence as shown in SEQ ID No: 1.

[0029] The application of the above-mentioned biomaterial in any of the following c1)-c4) should also be within the protection scope of this application: c1) Preparation of products for the treatment or adjunctive treatment of Ebola virus disease; c2) Preparation of products that inhibit Ebola virus infection; c3) Preparation of products that inhibit the proliferation of Ebola virus; c4) Preparation of products for the prevention of Ebola virus disease.

[0030] The present application uses the above-mentioned type of reagents for inhibiting the expression of the methyltransferase 3 gene to inhibit the proliferation of Ebola virus or treat Ebola virus. Specifically, the use of the above-mentioned STM2457 compound or the methyltransferase 3 gene siRNA with a nucleotide sequence such as that shown in SEQ ID No: 1 can inhibit the m6A methylation level of Ebola virus and inhibit the proliferation of Ebola virus; it can also significantly inhibit the expression of Ebola virus protein VP40 and inhibit the formation of Ebola virus inclusion bodies.

[0031] Compared with the prior art, this application has the following beneficial effects: The present application provides the use of methyltransferase 3 as a target for treating or inhibiting Ebola virus. The present application finds that the use of methyltransferase 3 gene siRNA can interfere with the expression of methyltransferase 3 gene and inhibit the proliferation of Ebola virus. In addition, the use of methyltransferase 3 gene inhibitor STM2457 can also significantly inhibit the proliferation of the virus, inhibit the methylation of the virus, inhibit the expression of viral VP40 protein, and inhibit the formation of viral inclusion bodies (IBs), indicating that the proliferation of Ebola virus can be inhibited by inhibiting the methyltransferase 3 gene, suggesting that the methyltransferase 3 gene can be used as a target for the treatment of Ebola virus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The result of transfecting siRNA of methyltransferase 3 gene in HEK293 cells in Example 1 (wherein, Figure 1 A is the result of measuring luciferase level in harvested cells after 24 hours. Figure 1 B is the result of WB detection of the expression level of transfected methyltransferase 3 gene. Figure 1 C is the result of grayscale scanning and comparison of expression levels of transfected methyltransferase 3 gene using ImageJ software); Figure 2 The results of the treatment of HEK293 cells with different drugs on the luciferase level and m6A methylation level in Example 2 are shown in FIG. Figure 2 A is the result of measuring the luciferase level in the harvested cells after 24 hours of treatment. Figure 2 B is the m6A methylation level of Ebola virus after 24 hours of treatment); Figure 3 The expression results of the bora virus protein VP40 in HEK293 cells treated with different drugs in Example 3 ( Figure 3 A is the expression level of viral protein VP40 in WB, Figure 3 B is the use of ImageJ software Figure 3 Grayscale scanning of vp40 in A to compare expression levels) Figure 4The results of the formation of Ebola virus inclusion bodies in HEK293 cells treated with different drugs in Example 4 ( Figure 4 A shows the size of viral inclusion bodies (IBs) observed under a confocal microscope using VP35 immunofluorescence staining. Figure 4 B is the statistical result of inclusion body area size using ImageJ software). DETAILED DESCRIPTION

[0033] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0035] The cell lines and plasmids involved in the following embodiments: HEK293 cells were purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences; plasmids pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, pCAGGS-T7, pCAGGS-Tim1 and p4cis-vRNA-Luc are disclosed in the literature Hoenen T, et al. Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes (J. Vis. Exp., 2014), and the public can obtain them from the Academy of Military Medical Sciences for repeating this experiment.

[0036] The molecular biological reagents and antibodies involved in the following examples: the transfection reagent Lipofectamine 3000 (L3000-015) was purchased from Thermo Company; opti-MEM was purchased from GIBCO.

[0037] The nucleotide sequence corresponding to the methyltransferase 3 gene (GeneBank: 56339) in the following examples is shown in SEQ ID No: 2, and the amino acid sequence of the methyltransferase 3 protein encoded by it is shown in SEQ ID No: 3 (NP_062826.2).

[0038] Nucleotide sequence:

[0039] Amino Acid Sequence: .

[0040] Example 1 EBOV minimal genome system: This study used the EBOV minimal genome system that can be operated in a biosafety level 2 laboratory to detect the virus level in cells by luciferase activity. The experimental operation process is briefly as follows: HEK293 cells were passaged into six-well plates (the confluence of cells after attachment was about 50%). After the cells were attached, Lipofectamine 3000 transfection reagent was used for transfection according to the instructions. siRNA of methyltransferase 3 gene (nucleotide sequence is 5'-CTGCAAGTATGTTCACTATGA-3', nucleotide sequence is shown in SEQ ID No: 1) was transfected into HEK293 cells. 24 hours after transfection, plasmids related to the EBOV minimal genome system were transfected: pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), p4cis-vRNA-Rluc (250ng) and pCAGGS-T7 (250ng). The operation method is briefly as follows: Dilute about 5μg siRNA to be transfected with 100μl opti-MEM; dilute 10μl Lipofectamine3000 with 100μl opti-MEM, add the diluted siRNA mixture dropwise to the diluted Lipofectamine3000 and mix well; leave at room temperature for 15min, and add the siRNA-liposome mixture dropwise to the cell culture medium. After 24 hours, dilute the plasmid to be transfected and 4μl P3000 with 100μl opti-MEM; dilute 3μl Lipofectamine3000 with 100μl opti-MEM, add the diluted plasmid and P3000 mixture dropwise to the diluted Lipofectamine3000 and mix well; leave at room temperature for 15min, and add the plasmid-liposome mixture dropwise to the cell culture medium.

[0041] Luciferase level experiment: Take the cells out of the incubator, discard the culture medium, and follow the instructions of the luciferase detection kit: Wash the cells with PBS, add 500μl PLB lysis buffer and lyse on a shaker for 15 minutes. After centrifugation, take 20μl of cell lysis supernatant and add it to 100μl Luciferase Assay Reagent Ⅱ. After mixing, put it into the TD-20 / 20 fluorescence photometer to measure the luminescence value RLU1, and then add 100μl Stop&Glo Reagent to measure the fluorescence luminescence value RLU2. The relative luciferase activity value of RLU2 / RLU1 evaluates the virus level, and then confirms the effect of siRNA knockdown or drug treatment on the proliferation of EBOV trVLP in cells.

[0042] like Figure 1 As shown, luciferase levels were measured in harvested cells 24 h later ( Figure 1A, Luciferase level), WB detection of the expression level of transfected methyltransferase 3 gene ( Figure 1 B) and using ImageJ software Figure 2 Grayscale scanning of the transfected methyltransferase 3 gene in B was performed to compare the expression levels ( Figure 1 C). The present application uses specific siRNA to interfere with the expression level of methyltransferase 3 gene in cells, which can significantly inhibit the proliferation of viruses.

[0043] Example 2 The EBOV minimal genome system-related plasmids were transfected into HEK293 cells: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng), and the operation method was the same as in Example 1.

[0044] After 24 hours, different concentrations of STM2457 (0μM, 1μM, 5μM, 10μM) were added for treatment (DMSO is the drug solvent and serves as a negative control), and another group was treated with the antiviral drug Remdesivir (as a positive control).

[0045] The results are as follows Figure 2 As shown, luciferase levels were measured in cells after 24 h of treatment ( Figure 2 A, Luciferase level), and m6A methylation level of Ebola virus ( Figure 2 B). The use of the methyltransferase 3 inhibitor STM2457 can significantly inhibit the m6A methylation level of Ebola virus and inhibit the proliferation of Ebola virus.

[0046] Example 3 The EBOV minimal genome system-related plasmids were transfected into HEK293 cells: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng), and the operation method was the same as in Example 1.

[0047] After 24 hours, STM2457 was added to the cells (DMSO is the drug solvent and serves as a negative control), and another group was treated with the antiviral drug Remdesivir (as a positive control).

[0048] like Figure 3As shown, cells were harvested after 24 hours of treatment, and the expression level of viral protein VP40 was measured by WB ( Figure 3 A), using ImageJ software Figure 3 The grayscale scan of vp40 in A was used to compare the expression levels ( Figure 3 B).

[0049] Example 4 The EBOV minimal genome system-related plasmids were transfected into HEK293 cells: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng) and pCAGGS-T7 (250 ng), and the operation method was the same as in Example 1.

[0050] After 24 hours, STM2457 was added to the cells (DMSO is the drug solvent and serves as a negative control), and another group was treated with the antiviral drug Remdesivir (as a positive control).

[0051] VP35 immunofluorescence staining experiment: Wash the cells three times with PBS, add 2 ml 4% paraformaldehyde, and let stand at room temperature for 30 minutes. After fixation, absorb the 4% paraformaldehyde and transfer it to the waste bottle; add 2 ml PBS, wash on a shaker for 5 minutes, and wash 3 times with PBS; add 1 ml 0.3% TritonX-100 (prepared in PBS) to perforate for 15 minutes, and wash 3 times with PBS; use 1 ml 5% goat serum blocking solution (prepared in PBS), block on a shaker for 30 minutes, and wash 3 times with PBS; prepare the primary antibody diluent (antibody: goat serum blocking solution = 1:50), add 100μl to each well, incubate at 4°C for 2 hours, and wash 3 times with PBS; prepare the secondary antibody diluent (antibody: goat serum blocking solution = 1:100), add 100μl to each well, incubate at room temperature for 1 hour, and wash 3 times with PBS. Finally, add a drop of DAPI-containing mounting medium and take pictures under a laser confocal microscope.

[0052] like Figure 4 As shown, cells were collected 24 hours after treatment and VP35 immunofluorescence staining was performed. The size of viral inclusion bodies (IBs) was observed under a confocal microscope ( Figure 4 A) and used ImageJ software to Figure 4 The size of the inclusion bodies in A was counted ( Figure 4 B).

[0053] As can be seen from the above content, the present application uses specific siRNA to interfere with the expression level of the methyltransferase 3 gene in cells, which can significantly inhibit the proliferation of the virus. The use of the methyltransferase 3 inhibitor STM2457 can significantly inhibit the m6A methylation level of Ebola virus and inhibit the proliferation of Ebola virus. The use of the methyltransferase 3 inhibitor STM2457 can significantly inhibit the expression of the Ebola virus protein VP40. The use of the methyltransferase 3 inhibitor STM2457 can significantly inhibit the formation of Ebola virus inclusion bodies. It shows that methyltransferase 3 can be used as a target to inhibit the proliferation of Ebola virus or treat Ebola virus, which has important application value for the treatment of Ebola virus disease.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. Use of an agent for knocking out the methyltransferase 3 gene or an agent for inhibiting the expression of the methyltransferase 3 gene in any one of the following a1) to a4), characterized in that: a1) Preparation of products for the treatment or adjunctive treatment of Ebola virus disease; a2) Preparation of products that inhibit Ebola virus infection; a3) Preparation of products that inhibit the proliferation of Ebola virus; a4) Preparation of products for the prevention of Ebola virus disease.

2. The use according to claim 1, characterized in that The reagent for inhibiting the expression of methyltransferase 3 gene includes methyltransferase 3 gene inhibitor or methyltransferase 3 gene siRNA.

3. The use according to claim 2, characterized in that The methyltransferase 3 gene inhibitor includes the STM2457 compound.

4. The use according to claim 3, characterized in that The concentration of the STM2457 compound was 1-10 μM.

5. The use according to claim 2, characterized in that The methyltransferase 3 gene siRNA is an siRNA with a nucleotide sequence as shown in SEQ ID No:

1.

6. Use of an agent for inhibiting the activity of methyltransferase 3 protein or an agent for reducing the content of methyltransferase 3 protein in any of the following b1) to b4), characterized in that: b1) Preparation of products for the treatment or adjunctive treatment of Ebola virus disease; b2) Preparation of products that inhibit Ebola virus infection; b3) Preparation of products that inhibit the proliferation of Ebola virus; b4) Preparation of products for the prevention of Ebola virus disease.

7. The use according to claim 6, wherein the agent for inhibiting the activity of methyltransferase 3 protein or the agent for reducing the content of methyltransferase 3 protein is siRNA or a methyltransferase 3 gene inhibitor having a nucleotide sequence as shown in SEQ ID No:

1.

8. Use of a methyltransferase 3 gene inhibitor in the preparation of a drug for inhibiting Ebola virus inclusion body formation, characterized in that: The methyltransferase 3 gene inhibitor includes the STM2457 compound.

9. Use of a methyltransferase 3 gene inhibitor in the preparation of a reagent for inhibiting the m6A methylation level of Ebola virus, characterized in that: The methyltransferase 3 gene inhibitor includes the STM2457 compound.

10. A biomaterial, characterized in that: The biological material includes an agent for inhibiting the expression of the methyltransferase 3 gene, and the agent for inhibiting the expression of the methyltransferase 3 gene includes an STM2457 compound or a methyltransferase 3 gene siRNA with a nucleotide sequence as shown in SEQ ID No: 1.

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