D8L protein-based monkey pox drug target and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a monkey pox target based on D8L protein and application of the monkey pox target, and the amino acid sequence of the D8L protein is shown as SEQ ID NO.1. After the relevance of the D8L protein on the inhibition effect of the interferon conduction pathway is detected, the application of the D8L protein as a monkey pox disease drug target is provided, and an antagonist for preparing a drug for preventing or treating the monkey pox disease is provided on the basis of the drug target.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to monkeypox disease targets based on D8L protein and their applications. Background Art

[0002] As of June 3, 2024, the Centers for Disease Control and Prevention in the United States has reported a total of 95,912 cases of monkeypox virus infection. Monkeypox virus is a zoonotic disease with symptoms including fever, severe headache, lymphadenopathy and myalgia. Currently, due to the surge in monkeypox cases in parts of Africa and the detection of the spread of new strains of monkeypox virus, the World Health Organization has announced on August 14, 2024 that "the monkeypox outbreak constitutes a public health emergency of international concern."

[0003] Monkeypox virus belongs to the genus Orthopoxvirus of the Poxviridae family. The genome of monkeypox virus is about 197205bp long and encodes about 198 proteins. The genome consists of a central conserved region and two variable regions. The central conserved region of monkeypox virus mainly encodes important enzymes such as DNA polymerase and RNA polymerase and viral structural proteins. The variable region encodes proteins related to the virulence of the virus itself and the regulation of host immune response.

[0004] At present, there is no specific treatment for the monkeypox virus epidemic, so researchers should focus on exploring new prevention policies and treatments. In the early stage of viral infection, the interferon (IFN) response produced by the host ISGs (Cig5, ISG56, OAS1, etc.) genes plays an important role in eliminating viral infection. However, monkeypox virus has evolved a host immune escape mechanism. According to relevant research reports, monkeypox virus can regulate the activation of host interferon and downstream antiviral signaling pathways through certain biomacromolecules to antagonize the host antiviral effect. Such biomacromolecules can provide a scientific basis for the development of monkeypox virus vaccines and the screening of antiviral drugs. The relevant gene of D8L protein is located in the variable region. This protein is closely related to the regulation of the host interferon signal transduction pathway. However, there is currently no relevant research on the D8L protein antagonizing the host immune escape strategy, nor is there a relevant target as a drug target. Summary of the invention

[0005] The problem that the present application aims to solve is, after ascertaining the relevance of the D8L protein to the inhibitory effect on the interferon conduction pathway, to provide the use of the D8L protein as a drug target for monkeypox disease, and based on the drug target, to provide an antagonist for the preparation of a preventive or therapeutic drug for monkeypox disease.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] On the one hand, the present application provides the use of D8L protein as a monkeypox drug target, and the amino acid sequence of the D8L protein is shown in SEQ ID NO.1.

[0008] In one aspect, the present application provides an antagonist of D8L protein, wherein the antagonist is designed with D8L protein or a gene related to D8L protein as a target.

[0009] Furthermore, the antagonist comprises siRNA.

[0010] Further, the siRNA includes one of siRNA1 and siRNA2;

[0011] Wherein, the siRNA1 includes siRNA1-F and siRNA1-R, and the srRNA2 includes siRNA2-F and siRNA2-R;

[0012] The nucleotide sequence of the siRNA1-F is shown in SEQ ID NO.2, the nucleotide sequence of the siRNA1-R is shown in SEQ ID NO.3, the nucleotide sequence of the siRNA2-F is shown in SEQ ID NO.4, and the nucleotide sequence of the siRNA2-R is shown in SEQ ID NO.5.

[0013] Furthermore, the antagonist is used to inhibit the transcription or expression of D8L protein-related genes.

[0014] On the one hand, the present application provides the use of D8L protein or D8L protein-related genes as research materials in studying the mechanism of monkeypox virus evading host immunity.

[0015] This application has the following beneficial effects:

[0016] (1) This application provides a new drug target for monkeypox disease by studying the escape mechanism of monkeypox virus from innate immunity, which has great potential in the prevention, treatment and drug screening of monkeypox disease.

[0017] (2) This application clarifies that the monkeypox virus D8L protein has an inhibitory effect on the interferon transduction pathway, determines its site of action, and reveals the molecular mechanism by which it exerts its inhibitory effect.

[0018] (3) The present application designs a siRNA based on the gene related to the D8L protein, which can restore the interferon signaling pathway inhibited by the D8L protein, demonstrating the feasibility of designing a drug for the prevention or treatment of monkeypox virus based on the drug target of the D8L protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present disclosure, the drawings required for use in some embodiments of the present disclosure are briefly introduced below.

[0020] Figure 1 The expression of D8L gene in experimental groups 1 to 3 in Example 2 of the present application in HEK-293T cells;

[0021] Figure 2 is the induction multiple of the interferon pathway under the influence of D8L protein in Example 2 of the present application;

[0022] Figure 3 The expression of IFITM1, IFIT1, ISG15, and STAT1 genes in Example 3 of the present application (A: control group 1; B: control group 2; C: control group 3; D: experimental group 1);

[0023] Figure 4 The transcription levels of Cig5, ISG56, and OAS1 genes under the influence of monkeypox virus D8L protein in Example 4 of the present application (A: Cig5; B: ISG56; C: OAS1);

[0024] Figure 5 The results of the positive pull and reverse pull experiments of the STAT1 gene under the influence of the D8L protein in Example 5 of the present application (Ⅰ and Ⅱ are positive pull experiments; Ⅲ and Ⅳ are reverse pull experiments; A: control group 1; B: control group 2; C: control group 3; D: experimental group 1; E: control group 4; F: control group 5; G: control group 6; H: experimental group 2);

[0025] Figure 6 The expression level of D8L gene inhibited by siRNA in Example 6 of the present application;

[0026] Figure 7 These are the ISGs transcription levels (A: Cig5; B: ISG56; C: OAS1) after siRNA1 inhibited D8L gene expression in Example 7 of the present application. DETAILED DESCRIPTION

[0027] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0028] 1. Cells, plasmids, reagents, and other materials

[0029] 1. Cells: HEK-293T human embryonic kidney cell line was purchased from Fenghui Biotechnology Co., Ltd. and preserved by the College of Medical Technology of Chengdu University of Traditional Chinese Medicine.

[0030] 2. Plasmids and siRNA: The eukaryotic expression plasmid of monkeypox virus D8L protein was designed by our laboratory and synthesized by Suzhou Hongxun Biotechnology Co., Ltd.; ISRE-luc, phRluc-TK, and IFN-λ eukaryotic expression plasmids were donated by R. Hartmann's team at Aarhus University; STAT1 plasmid was purchased from Miaoling Biotechnology; two pairs of small interfering RNA (siRNA) were designed by our laboratory and synthesized by Sangon Biotechnology Co., Ltd.

[0031] 3. Reagents: PEI transfection reagent was purchased from Fushen Biotechnology Co., Ltd., Lipofectamine 3000 transfection reagent was purchased from Thermo Fisher Scientific, Flag tag antibody and STAT1 antibody were purchased from Wuhan Sanying Company, IFITM1antibody, IFIT1antibody, pSTAT1 antibody, and ISG15 antibody were purchased from Abmart Company, and Anti-EGFPNanbodyAgarose Beads and Anti-Flag NanbodyAgarose Beads were purchased from Kangti Life Science Company.

[0032] 4. DMEM cell culture medium was a product of Wuhan Pronocell Life Science Co., Ltd., and was supplemented with 10% fetal bovine serum purchased from Thermo Fisher, USA, non-essential amino acids, ampicillin and streptomycin (100 U / ml each), and the culture medium additives were all products of Beijing Solaibao Technology Co., Ltd.

[0033] 5. Cell digestion solution, containing 0.25% trypsin, was purchased from Cytiva, USA.

[0034] 6. Western and IP cell lysates were purchased from Abbkine Scientific Co., Ltd.

[0035] 7. Loading buffer: Omni-Easy TM The instant protein loading buffer (denatured, reduced, 5×) was purchased from Yazyme Corporation.

[0036] 8.PBS: PBS washing buffer (20×), ARO030-1, Boster.

[0037] 9.TWEEN 20:Shanghai Bioengineering Co., Ltd.

[0038] 2. Experimental methods:

[0039] Example 1: Construction of eukaryotic expression plasmid for D8L protein

[0040] (1) The gene sequence of the monkeypox virus D8L protein was downloaded from NCBI, as shown in SEQ ID NO.6. The codons were optimized. According to the correspondence between codons and amino acids, the amino acid sequence of the D8L protein was shown in SEQ ID NO.1. Then, mCherry and Flag tags were connected to the C-terminus and constructed into the PcDNA3.1+ vector. Then, it was sent to Suzhou Hongxun Biotechnology Co., Ltd. for synthesis to obtain the eukaryotic expression plasmid of the monkeypox virus D8L protein.

[0041] Example 2 verifies the effect of monkeypox virus D8L protein on interferon signal transduction.

[0042] (1) HEK-293T cells were cultured in DMEM cell culture medium;

[0043] (2) The cultured HEK-293T cells were cultured at 6×10 5 / well inoculated into 6-well plates;

[0044] (3) After 24 h, 5 wells were selected and the HEK-293T cells in the wells were co-transfected with plasmids according to the dosage in Table 1. Among them, the quality control plasmid phRluc-TK was inoculated to express Renilla luciferase, and the transfection efficiency of each experimental group was normalized;

[0045] Table 1 Plasmid dosage for the experiment on the effect of D8L protein on interferon signal transduction

[0046]

[0047] (4) 24 h after co-transfection, 200 μL of Western and IP lysis buffer was added to lyse the cells, and the expression of mCherry protein in experimental groups 1 to 3 was observed under a fluorescence microscope ( Figure 1 ), and the dual luciferase activity detection kit was used to detect the firefly luciferase activity and the sea renilla luciferase activity. The detection value of firefly luciferase was compared with the detection value of sea renilla luciferase to reduce the influence of internal variation factors on the accuracy of the experiment, that is, the transfection efficiency of each well was standardized, and then the luciferase detection values ​​of the control groups 1 to 2 and the experimental groups 1 to 3 were divided by the luciferase detection value of the control group 1, so as to obtain the interferon pathway induction fold of each group ( Figure 2 ).

[0048] The results showed that both the monkeypox virus D8L plasmid and IFN-λ could be correctly expressed in HEK-293T cells, and the monkeypox virus D8L protein could significantly inhibit the interferon signaling pathway, indicating that the D8L protein is one of the virulence factors that enables monkeypox virus to evade innate immunity.

[0049] Example 3 Verification of the effect of monkeypox virus D8L protein on the expression level of interferon-stimulated genes (ISGs)

[0050] (1) HEK-293T cells were cultured in DMEM cell culture medium;

[0051] (2) The cultured HEK-293T cells were cultured at 6×10 5 / well inoculated into 6-well plates;

[0052] (3) 24 h later, four wells were selected and the HEK-293T cells in the wells were co-transfected with plasmids according to the dosages in Table 2;

[0053] Table 2 Plasmid dosage for the experiment on the effect of D8L protein on the expression level of ISGs protein

[0054]

[0055] (4) After plasmid transfection, the cells were cultured in a cell culture incubator for 24 h;

[0056] (5) After the culture is completed, 200 μL of Western and IP lysis buffer was added to each well after transfection to lyse the cells. After protein samples were extracted, loading buffer was added to prepare WB samples. Two replicate groups were set up for the above experiment;

[0057] (6) The expression of IFITM1, IFIT1, ISG15, and STAT1 genes in the WB samples prepared from control groups 1 to 3 and experimental group 1 was detected by WB experiment ( Figure 3 ).

[0058] The results showed that the monkeypox virus D8L protein could significantly inhibit the expression of ISGs (IFITM1, IFIT1, ISG15, STAT1) proteins, indicating that the monkeypox virus D8L protein could inhibit the interferon signal transduction pathway, and the D8L protein had the most obvious inhibitory effect on STAT1. Therefore, the mechanism by which monkeypox virus evades innate immunity is related to the interaction between the D8L protein and the STAT1 gene.

[0059] Example 4 Verification of the effect of D8L protein on ISGs mRNA transcription level

[0060] (1) HEK-293T cells were cultured in DMEM cell culture medium;

[0061] (2) Cultured HEK-293 cells were plated at 6×10 5 / well inoculated into 6-well plates;

[0062] (3) 24 h later, four wells were selected and the HEK-293T cells in the wells were co-transfected with plasmids according to the dosages in Table 3;

[0063] Table 3 Plasmid dosage for the experiment on the effect of D8L protein on ISGs mRNA transcription level

[0064]

[0065] (4) After plasmid transfection, the cells were cultured in a cell culture incubator for 24 h;

[0066] (5) After the culture was completed, 200 μL of Western and IP lysis buffer was added to each well after transfection to lyse the cells, extract the cellular RNA, reverse transcribe the RNA into DNA, and perform RT-QPCR reaction on the reverse transcribed DNA to detect and record the levels of Cig5, ISG56, and OAS1 gene transcription into mRNA ( Figure 4 ).

[0067] The results showed that the monkeypox virus D8L protein had an inhibitory effect on the transcription of ISGs (Cig5, ISG56, OAS1) genes into mRNA, among which the inhibitory effect on the transcription level of Cig5 and OAS1 genes was the most significant.

[0068] Example 5 Verification of whether monkeypox D8L protein can interact with STAT1

[0069] (1) HEK-293T cells were cultured in DMEM cell culture medium;

[0070] (2) The cultured HEK-293T cells were cultured at 6×10 5 / well was inoculated into two 6-well plates;

[0071] (3) 24 hours later, 1 μg of empty vector plasmid and 1 μg of STAT1 plasmid were transfected into the cells in the No. 1 6-well plate, and 1 μg of eukaryotic expression plasmid of monkeypox virus D8L protein and 1 μg of STAT1 plasmid were transfected into the cells in the No. 2 6-well plate;

[0072] (4) After plasmid transfection, the cells were cultured in a cell culture incubator for 24 h, and 200 μL of lysis buffer was added to each well of two 6-well plates to lyse the cells, and then CO-IP forward and reverse pull experiments were performed;

[0073] (5) CO-IP positive pull-up experiment: Take 50 μL of protein sample in 6-well plate No. 1 as control group 1, take 50 μL of protein sample in 6-well plate No. 2 as control group 2, and take another 150 μL of protein sample in 6-well plate No. 1 as control group 3, and take another 150 μL of protein sample in 6-well plate No. 2 as experimental group 1. The control group 3 and experimental group 1 were incubated with 25 μL of nanobeads carrying Flag antibodies after equilibrium overnight, and then washed three times with 1×PBST+0.05% TWEEN 20, eluted with 1×Loading buffer and detected by WB;

[0074] (6) CO-IP reverse pull-down experiment: Take 50 μL of protein sample in 6-well plate No. 1 as control group 4, take 50 μL of protein sample in 6-well plate No. 2 as control group 5, take another 150 μL of protein sample in 6-well plate No. 1 as control group 6, take another 150 μL of protein sample in 6-well plate No. 2 as experimental group 2, incubate control group 6 and experimental group 2 with equilibrated 25 μL of nanobeads carrying EGFP antibody overnight, then wash with PBST three times, add 1× Loading buffer for elution and perform WB detection;

[0075] (7) Design two sets of repetitions for the CO-IP forward and reverse pull experiments, respectively, and observe and record the results of the forward and reverse pull experiments. Figure 5 ).

[0076] The forward and reverse pull results of CO-IP show that the D8L protein of monkeypox virus can interact with the STAT1 protein in the interferon signaling pathway, indicating that the D8L protein achieves the effect of escaping the body's immunity by interacting with the STAT1 protein.

[0077] Example 6 Screening of siRNA targeting the D8L gene and verifying its ability to inhibit the expression level of the D8L gene

[0078] (1) Two pairs of siRNAs were designed based on the gene sequence of D8L protein, and were named siRNA1 and siRNA2, respectively. siRNA1 included siRNA1-F and siRNA1-R, the nucleotide sequence of siRNA1-F was shown in SEQ ID NO.2, and the nucleotide sequence of siRNA1-R was shown in SEQ ID NO.3. siRNA2 included siRNA2-F and siRNA2-R, the nucleotide sequence of siRNA2-F was shown in SEQ ID NO.4, and the nucleotide sequence of siRNA2-R was shown in SEQ ID NO.5.

[0079] (2) HEK-293T cells were cultured in DMEM cell culture medium;

[0080] (3) The cultured HEK-293T cells were cultured at 6×10 5 / well inoculated into 6-well plates;

[0081] (4) After 24 h, three wells were selected and the HEK-293T cells in the wells were co-transfected with plasmids and siRNA according to the dosages in Table 4;

[0082] Table 4 Plasmid dosage for different siRNAs to inhibit monkeypox virus D8L gene expression experiments;

[0083]

[0084] (5) After transfection of plasmid and siRNA, the cells were cultured in a cell culture incubator for 24 h;

[0085] (6) Cell RNA was extracted from experimental groups 1 to 2 and control group 1, reverse transcribed into DNA, and RT-QPCR reaction was performed to detect and record the level of D8L gene transcription into mRNA ( Figure 6 ).

[0086] The results showed that both siRNA1 and siRNA2 could effectively inhibit the expression of D8L gene, among which siRNA1 had the best effect in inhibiting the transcription of D8L gene into mRNA.

[0087] Example 7 Verification of ISGs transcription levels after siRNA1 inhibits D8L gene expression

[0088] (1) HEK-293T cells were cultured in DMEM cell culture medium for 24 h;

[0089] (2) The cultured HEK-293T cells were cultured at 6×10 5 / well inoculated into 6-well plates;

[0090] (3) 24 hours later, 0.5 μg of eukaryotic expression plasmid of monkeypox virus D8L protein, 0.5 μg of eukaryotic expression plasmid of IFN and 50 pmoles of siRNA1 were transfected into cells in three wells as the experimental group, and 0.5 μg of eukaryotic expression plasmid of monkeypox virus D8L protein and 0.5 μg of eukaryotic expression plasmid of IFN were transfected into cells in the other three wells as the control group;

[0091] (4) After transfection of plasmid and siRNA, the cells were cultured in a cell culture incubator for 24 h;

[0092] (5) Cell RNA was extracted from the experimental group and the control group, and then reverse transcribed into DNA for RT-QPCR reaction to detect and record the mRNA transcription levels of Cig5, ISG56, and OAS1 ( Figure 7 ).

[0093] The results showed that after siRNA1 inhibited the expression of D8L gene, the transcription level of ISGs was significantly restored, among which the transcription level of ISG56 recovered the highest degree, indicating that siRNA1 can effectively inhibit the effect of monkeypox virus on the transcription level of ISGs and has the potential to be used as a drug for the initial screening of monkeypox disease.

[0094] In summary, the present application verifies that the D8L protein can significantly inhibit the function of the interferon signaling pathway. The specific mechanism is that the D8L protein interacts with STAT1 in the pathway, thereby inhibiting the transcription or expression of ISGs genes. Therefore, the monkeypox virus D8L protein is one of the virulence factors of the monkeypox virus and can be used as a drug target for anti-monkeypox virus infection. The siRNA initially screened out based on the D8L protein has the ability to inhibit the transcription of the monkeypox virus D8L gene, thereby inhibiting the escape of the monkeypox virus from innate immunity, and can be used to prepare preventive or therapeutic drugs for monkeypox virus in the future.

[0095] The above is only a preferred embodiment of the present disclosure. It should be understood that the present disclosure is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present disclosure, and should be within the scope of protection of the claims attached to the present disclosure.

Claims

1. The use of D8L protein as a monkeypox drug target, characterized in that: The amino acid sequence of the D8L protein is shown in SEQ ID NO.

1.

2. An antagonist of D8L protein, characterized in that The antagonist is designed with D8L protein or a gene related to D8L protein as a target.

3. The antagonist according to claim 2, characterized in that The antagonists include siRNA.

4. The antagonist according to claim 3, characterized in that The siRNA includes one of siRNA1 and srRNA2; Wherein, the siRNA1 includes siRNA1-F and siRNA1-R, and the srRNA2 includes siRNA2-F and siRNA2-R; The nucleotide sequence of the siRNA1-F is shown in SEQ ID NO.2, the nucleotide sequence of the siRNA1-R is shown in SEQ ID NO.3, the nucleotide sequence of the siRNA2-F is shown in SEQ ID NO.4, and the nucleotide sequence of the siRNA2-R is shown in SEQ ID NO.

5.

5. The antagonist according to any one of claims 2 to 4, characterized in that The antagonist is used to inhibit the transcription or expression of D8L protein related genes.

6. Application of D8L protein or D8L protein-related genes as research materials in studying the mechanism of monkeypox virus evading host immunity.

Images