Application of substance targeting EGLN1-HIF1alpha signal axis in preparation of medicine for treating Ebola virus infection

Abstract

The invention discloses application of a substance targeting an EGLN1-HIF1alpha signal axis in preparation of a medicine for treating Ebola virus infection in the technical field of biological medicine, and aims to solve the problems that the EGLN1-HIF1alpha signal axis has a function blank in Ebola virus replication and the interaction between VP35 and the EGLN1-HIF1alpha signal axis is unknown. Experiments prove that Ebola virus replication can be promoted through EGLN1 knockout or inhibition, Ebola virus proliferation is inhibited through EGLN1 overexpression, and Ebola virus proliferation is remarkably inhibited through HIF1alpha knockout or inhibition. The invention clarifies the effect of an EGLN1-HIF1alpha signal axis in Ebola virus replication, and provides a novel Ebola virus inhibitor targeting the signal axis and an application scheme.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of substances targeting the EGLN1-HIF1α signaling axis in the preparation of drugs for treating Ebola virus infection. Background Technology

[0002] Ebola virus (EBOV) belongs to the Filoviridae family and is a single-stranded negative-sense RNA virus. Its genes encode seven structural proteins, including nucleoprotein (NP), VP35, and VP40. VP35 is a key cofactor in viral RNA synthesis and also antagonizes type I interferon responses, playing a central role in viral replication and immune evasion. During viral replication, RNA forms ribonucleoprotein complexes with NP, VP35, etc., which aggregate in inclusion bodies. Inclusion bodies are the key sites for viral replication and proliferation.

[0003] The Egl-9 family hypoxia-inducible factor 1 (EGLN1), also known as PHD2 (ProlylHydroxylase Domain 2), is a core oxygen-sensing prolyl hydroxylase that catalyzes the hydroxylation of hypoxia-inducible factor 1 alpha (HIF1α) under normoxic conditions, leading to its ubiquitination and degradation. HIF1α, as a transcription factor, regulates the expression of hypoxia-related genes, metabolic and immune-related genes, and participates in viral replication regulation in various viral infections. The EGLN1-HIF1α signaling axis has been confirmed to be involved in the replication process of various viruses, including human cytomegalovirus and SARS-CoV-2, but its function and regulatory mechanism in Ebola virus infection are completely unknown.

[0004] VP35 is a key virulence protein of Ebola virus, and its known functions are concentrated in interferon antagonism and viral RNA synthesis. Previous studies have found that it can interact with a variety of host proteins, but its interaction with the host oxygen sensing pathway has not yet been revealed. In addition, there are no reports on whether VP35 affects viral replication by regulating the EGLN1-HIF1α signaling axis.

[0005] Currently, there is a lack of antiviral strategies targeting the VP35-EGLN1-HIF1α regulatory pathway of Ebola virus. Existing drugs often target viral entry or RNA polymerase, resulting in single-target drugs. There is a need to develop novel antiviral drugs based on host factors. Summary of the Invention

[0006] The purpose of this invention is to address the functional gaps in the EGLN1-HIF1α signaling axis during Ebola virus replication and the unknown interaction between VP35 and this signaling axis. This invention proposes the application of substances targeting the EGLN1-HIF1α signaling axis in the preparation of drugs for treating Ebola virus infection, clarifies the role of the EGLN1-HIF1α signaling axis in Ebola virus replication, and provides novel Ebola virus inhibitors targeting this signaling axis and their application strategies.

[0007] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the invention, the use of a substance targeting the EGLN1-HIF1α signaling axis in the preparation of a drug for treating Ebola virus infection is provided.

[0008] As a further optimization of the present invention, the substance targeting the EGLN1-HIF1α signal axis has any one of the following functions (1)-(2): (1) Promotes the expression level of the EGLN1 gene; (2) Interfere with or reduce the expression level of the HIF1α gene.

[0009] As a further optimization of the present invention, the substance targeting the EGLN1-HIF1α signaling axis promotes the expression level of the EGLN1 gene, thereby inhibiting intracellular Ebola virus replication and inhibiting the expression level of the Ebola virus genome VP40 gene.

[0010] As a further optimization of the present invention, the substance targeting the EGLN1-HIF1α signal axis includes any one of the following (1)-(5): (1) EGLN1 agonists; (2) Overexpression plasmid of EGLN1 gene; (3) HIF1α inhibitors; (4) HIF1α siRNA; (5) Gene editing tools that knock out HIF1α gene expression.

[0011] As a further optimization of the present invention, the overexpression plasmid of the EGLN1 gene is constructed by inserting the CDS sequence of the EGLN1 gene into the pcdna3.1(+) plasmid.

[0012] As a further optimization of the present invention, the HIF1α inhibitor is either BAY 87-2243 or PX-478 2HCl.

[0013] As a further optimization of the present invention, the HIF1α siRNA is an siRNA formed by annealing two single-stranded molecules as shown in SEQ ID No. 1 and SEQ ID No. 2: SEQ ID No.1: 5'-CAACCUCAGUGUGGGUAUATT-3'; SEQ ID No. 2: 5'-UAUACCCACACUGAGGUUGTT-3'.

[0014] As a second aspect of the invention, a drug for treating Ebola virus infection is provided, the drug being used to interfere with or reduce the expression level of the HIF1α gene or promote the expression level of the EGLN1 gene.

[0015] As a further optimization of the present invention, the drug includes any one of the following (1)-(5): (1) EGLN1 agonists; (2) Overexpression plasmid of EGLN1 gene; (3) HIF1α inhibitors; (4) HIF1α siRNA; (5) Gene editing tools that knock out HIF1α gene expression.

[0016] As a third aspect of the present invention, a method for designing a drug for treating Ebola virus infection is provided, which is not for the purpose of disease treatment and diagnosis, but targets the EGLN1-HIF1α signaling axis to interfere with or reduce the expression of the HIF1α gene or promote the expression of the EGLN1 gene.

[0017] The beneficial effects of this invention are as follows: This invention reveals for the first time the interaction between Ebola virus VP35 and host EGLN1, and clarifies the regulatory mechanism of the EGLN1-HIF1α signaling axis in Ebola virus replication, filling a research gap in this field. This invention provides a dual-target antiviral strategy: it can provide a theoretical basis for the development of agonists targeting EGLN1, and can also directly apply HIF1α inhibitors (such as BAY and PX478) to inhibit Ebola virus replication, with flexible target selection. The substance targeting the host EGLN1-HIF1α signaling axis provided by this invention is less likely to induce drug resistance in the virus, thus solving the problem of drug resistance in traditional drugs that target the virus's own proteins. The experiments conducted in this invention are based on the trVLP system, which can be carried out in a biosafety level 2 laboratory, providing a safe and efficient research platform for subsequent drug development. Attached Figure Description

[0018] Figure 1 The plasmid map of pcdna3.1(+)-EGLN1-flag provided by this invention; Figure 2 This invention provides a comparison of the replication levels of Ebola virus P0 virus-like particles in EGLN1-KO cells and wild-type cells. Figure 3 Comparison of Ebola virus P1 virus-like particle replication levels between EGLN1-KO cells and wild-type cells provided by this invention; Figure 4 The invention provides the following: after overexpression of EGLN1-Flag, the replication level of P0 virus-like particles and the expression level of the viral genome VP40 gene in the supernatant; Figure 5 The invention provides the following data: after overexpression of EGLN1-Flag, the replication level of P1 virus-like particles and the expression level of the viral genome VP40 gene in the supernatant. Figure 6 The replication level of Ebola virus P0 virus-like particles in cells after treatment with the EGLN1 inhibitors DMOG, CoCl2, and IXO4 in a concentration gradient provided by the present invention; Figure 7 The results of Western blot detection of HIF1α protein levels provided by this invention; Figure 8 The detection results of HIF1α interference inhibiting P0 virus-like particles provided by the present invention; Figure 9 The detection results of the inhibition of P0 virus-like particles by the HIF1α inhibitors BAY 87-2243 and PX-478 2HCl provided by the present invention; Figure 10 The detection results of the inhibition of P1 virus-like particles by the HIF1α inhibitors BAY 87-2243 and PX-478 2HCl provided by the present invention. Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] I. Materials and Methods 1. trVLP system The Ebola virus (EBOV) transcription and replication-competent virus-like particle (trVLP) system used in this study includes plasmids such as pCAGGS-NP, pCAGGS-VP35, pCAGGS-VP30, pCAGGS-L, p4cis-vRNA-Rluc, pCAGGS-T7, and pCAGGS-Tim1. These plasmids were constructed based on the reported minigenome (MG) technology (Hoenen, T., Watt, A., Mora, A., Feldmann, H. Modeling The Lifecycle Of Ebola Virus UnderBiosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes. J. Vis. Exp.(91), e52381, doi:10.3791 / 52381(2014)., can simulate the complete life cycle of EBOV (transcription, replication, assembly and multiple rounds of infection) under biosafety level 2 (BSL-2) conditions, without the risk of generating live virus.

[0021] trVLP system core components (1) Mini genome plasmid (p4cis-vRNA-Rluc): Contains conserved promoter sequences at both ends of the EBOV genome, with the Renilla luciferase (Rluc) reporter gene and viral structural protein (VP40, GP, VP24) coding sequences inserted in the middle, used to monitor viral replication activity and simulate viral particle assembly; (2) Replication Transcription Core Complex (RNP) plasmid: including pCAGGS-NP (nuclear protein), pCAGGS-VP35 (RNA polymerase cofactor), pCAGGS-VP30 (transcription activator) and pCAGGS-L (RNA-dependent RNA polymerase), which provide essential enzymatic support for the transcription and replication of viral mini-genomes; (3) Helper plasmids: pCAGGS-T7 (provides T7 RNA polymerase to initiate mini-genome transcription) and pCAGGS-Tim1 (expresses EBOV cell invasion receptor Tim1 to optimize target cell infection efficiency). (4) Internal control plasmid: pGL4-Basic (firefly luciferase-encoded plasmid), used to correct transfection efficiency and cell state differences.

[0022] 1.2 Preparation and Infection Procedure of the trVLP System (1) Preparation and transfection of P0 generation cells: Human embryonic kidney HEK293T cells were transfected at a rate of 4 × 10⁻⁶ cells / year. 5 Each well was seeded with one plasmid and incubated at 37°C and 5% CO2 for 24 h until confluence reached 70%-80%. Following the Lipofectamine 3000 transfection reagent instructions, each well was co-transfected with the following plasmid mixture: pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), p4cis-vRNA-Rluc (250 ng), pCAGGS-T7 (250 ng), and pGL4-Basic internal control plasmid (25 ng), for a total plasmid amount of 4 μg. 4-6 h post-transfection, the medium was replaced with DMEM containing 5% fetal bovine serum (FBS), and the culture was continued for 48 h. The supernatant was collected, which is the P0 generation trVLP.

[0023] (2) P1 generation target cell infection and culture: HEK293T cells were infected and cultured at a rate of 4 × 10⁻⁶ cells / year. 5 Cells were seeded per well in 6-well plates and cultured for 24 h. Then, the cells were transfected with pCAGGS-NP (125 ng), pCAGGS-VP35 (125 ng), pCAGGS-VP30 (75 ng), pCAGGS-L (1000 ng), and pCAGGS-Tim1 (250 ng) plasmids to provide RNP complex and receptor support for trVLP replication after infection. 24 h after transfection, the target cell supernatant was discarded, and P0 generation trVLP supernatant (2 mL per well) was added. The cells were infected at 37°C for 24 h, then the medium was replaced with DMEM containing 5% FBS, and the cells were cultured for another 72 h. P1 generation cells and supernatant were collected for subsequent assays.

[0024] 2. Plasmids The PGL4-Basic plasmid was preserved by our research group and purchased from Promega. The pcdna3.1(+)-EGLN1-flag plasmid was constructed by our research group. It was obtained by inserting the CDS sequence of EGLN1 (NCBI Reference Sequence: NM_022051.3; CCDS: CCDS1595.1) into the pcdna3.1(+) plasmid. The image of the pcdna3.1(+)-EGLN1-flag plasmid (hereinafter referred to as EGLN1-flag plasmid) is shown below. Figure 1 As shown.

[0025] 3. Cells Human embryonic kidney HEK293T cells and EGLN1 knockout HEK293T cells (denoted as EGLN1-KO cells) were both constructed by Yuanjing Biotechnology and preserved in our laboratory. The above cells were cultured in DMEM medium (catalog number: C11965500BT) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and FBS (catalog number: 10099141C) were both Gibco products.

[0026] 4. Reagents The Dual Luciferase Assay Kit (catalog number: E1910) and the Renilla-Glo® Luciferase Assay Kit (catalog number: E2710) were both purchased from Promega. The transfection reagent was Lipfectamin 3000 (Invitrogen, catalog number: 2966989). Nucleic acid reagents were all purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.: siRNA targeting HIF1α (sense strand: 5'-CAACCUCAGUGUGGGUAUATT-3' (SEQ ID No. 1); antisense strand: 5'-UAUACCCACACUGAGGUUGTT-3' (SEQ ID No. 2)). Negative control siRNA (NC, sense strand: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No. 3); antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID No. 4)); HIF1α inhibitors: BAY 87-2243 (Selleck, catalog number: S7309) and PX-478 2HCl (Selleck, catalog number: S7612), purchased from Selleck. EGLN1 inhibitors DMOG (catalog number: HY-15893), CoCl2 (catalog number: S1233), and IXO4 (catalog number: HY-120110) were purchased from MedChemExpress. qPCR reagents: RNA extraction kit (Qiagen, catalog number: 74106), qPCR kit (promega A6020); qPCR primers: β-actin (internal reference): F-TGACGTGGACATCCGCAAAG (SEQ ID No. 5); R-CTGGAAGGTGGACAGCGAGG (SEQ ID No. 6); VP40 gene (viral target gene): VP40-F-5'-GGAGGCCATATACCCTGTCAGGTC-3' (SEQ ID No. 7); VP40-R-5'-GCCTGGTGTGTGGCTGGCAT-3' (SEQ ID No. 8).

[0027] 5. Western Blot (WB) Experimental Materials and Methods 5.1 Materials for Western Blot (WB) Experiment Western blot related antibodies: HIF1α antibody (Proteintech, catalog number: 66730-1-Ig); β-actin antibody (Proteintech, catalog number: HRP-60008); anti-EGLN1 antibody (ABIN702640, dilution ratio 1:1000); anti-Flag-HRP antibody (Sigma, catalog number: A8592, dilution ratio 1:5000); goat anti-rabbit IgG secondary antibody (HRP-labeled, catalog number: ZF-0316, dilution ratio 1:5000).

[0028] 5.2 Western Blot (WB) Experimental Method Western blotting (WB) was used to detect the expression levels of EGLN1, HIF1α, viral proteins, and internal reference proteins. The specific steps are as follows: (1) Collect the treated cells, wash twice with pre-cooled PBS, and add cell lysis buffer containing protease inhibitors (per 1×10⁻⁶ cells). 6 Cells were added to 100 μL and lysed on ice for 30 min, vortexed 3 times (10 s each time) during the process, centrifuged at 13400 rpm and 4℃ for 10 min, and the supernatant was used as the total protein sample. The protein concentration was determined by BCA method. (2) Mix the above total protein sample with 5× loading buffer at a ratio of 4:1, boil at 95°C for 10 min, cool in an ice bath and set aside. (3) Prepare SDS-PAGE gel (5% stacking gel, 10% separating gel), load 30 μg of protein per well, electrophore at 80V until the sample enters the separating gel, adjust the voltage to 120V, and continue electrophoresis for 90 min.

[0029] (4) Wet transfer: Align the gel with the PVDF membrane, place it in the transfer buffer for 10 min, put it in the transfer apparatus, transfer for 15 min, and ensure that there are no air bubbles between the gel and the membrane; (5) After the membrane transfer, the PVDF membrane is placed in the rapid sealing solution and sealed on a shaker at room temperature for 1 hour. The sealing solution is then recovered.

[0030] (6) Primary antibody incubation: Dilute the corresponding primary antibody (anti-HIF1α 1:1000, anti-EGLN1 1:1000, anti-β-actin 1:5000, anti-Flag 1:5000) with blocking solution and incubate overnight at 4°C on a shaker.

[0031] (7) Wash the membrane with TBST 3 times, 5 min each time; dilute the HRP-labeled secondary antibody (1:5000) with blocking solution, incubate on a shaker at room temperature for 1 h, wash the membrane with TBST 3 times again, 5 min each time, wipe off the residual liquid, spread the ECL chemiluminescent substrate evenly, and develop with a developer.

[0032] Unless otherwise specified, all materials used in this invention are commercially available products. Unless otherwise specified, all methods used below are conventional methods known to those skilled in the art.

[0033] II. Implementation Plan 1. Construction of a modified Ebola virus minimal genome system The modified Ebola virus minimal genome system used in this experiment can be used to study Ebola virus replication and infection in a biotechnology level 2 laboratory. The specific construction method is as follows: On day 1, HEK293T cells were seeded in 6-well plates at a density of 4 × 10⁶ cells per well. 5 indivual; On day 2 (24 hours later), each well was transfected with plasmids 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). On the third day, the cell culture supernatant was replaced with a culture medium containing 5% FBS. The resulting cell culture supernatant was the cell supernatant containing the P0 virus. On day 4, HEK293T cells (virus-targeting cells, or P1 cells for short) were seeded into 6-well plates at a density of 4 × 10⁶ cells per well. 5 indivual; On day 5, plasmids pCAGGS-NP (125ng), pCAGGS-VP35 (125ng), pCAGGS-VP30 (75ng), pCAGGS-L (1000ng), and pCAGGS-Tim1 (250ng) were transfected into P1 generation cells; On day 6, the supernatant was discarded, and then the P0 cell supernatant obtained on day 3 was added, and the infection was carried out for 24 hours. On day 7, replace the supernatant with a medium containing 5% FBS and continue culturing for 72 hours. Then collect the supernatant, which is the P1 cell supernatant. Continue to passage the cells according to the above steps or freeze them at -80°C.

[0034] 2. Experiment on the effect of EGLN1 on viral replication (2.1) Detection of P0 virus-like particle replication (wild-type vs EGLN1-KO cells) On day 1, HEK293T wild-type cells and EGLN1-KO cells were seeded into 6-well plates, 4 × 10⁴ cells per well. 5 Cells were placed in a 37°C, 5% CO2 incubator for 24 hours to serve as P0 virus-producing cells.

[0035] On day 2, the two types of P0-producing cells were transfected with the trVLP system plasmids in the following ratio: pCAGGS-NP (125ng) + pCAGGS-VP35 (125ng) + pCAGGS-VP30 (75ng) + pCAGGS-L (1000ng) + p4cis-vRNA-Rluc (250ng) + pCAGGS-T7 (250ng). At the same time, 10ng of PGL4-Basic internal control plasmid was added. The transfection was performed using Lipfectamin 3000 according to the instructions.

[0036] 24 hours after transfection, the medium was replaced with fresh medium containing 5% FBS, and the cells were cultured for another 48 hours to induce the production and release of P0 virus-like particles into the supernatant. Cells were collected, and intracellular luciferase activity was detected using a dual-luciferase assay kit and a multi-functional microplate reader to quantify the replication level of P0 virus-like particles in the two groups.

[0037] The results are as follows Figure 2 As shown, dual-luciferase assays revealed that, compared with wild-type cells, EGLN1-KO cells exhibited a significantly increased replication level of Ebola virus P0 virus-like particles.

[0038] (2.2) Detection of P1 virus-like particle replication (wild-type vs. P0 infection from EGLN1-KO cells) On day 4, HEK293T wild-type cells were seeded into new 6-well plates at a density of 4 × 10⁶ cells per well. 5 One cell was cultured for 24 hours and used as a P1 target cell.

[0039] On day 5, P1 target cells were transfected with pCAGGS-NP (125ng) + pCAGGS-VP35 (125ng) + pCAGGS-VP30 (75ng) + pCAGGS-L (1000ng) + pCAGGS-Tim1 (250ng) to prepare for P0 infection.

[0040] 24 hours after transfection, the supernatant of P1 target cells was discarded, and the supernatant of P0 virus-like particles produced by wild-type cells and EGLN1-KO cells as described in section 2.1 above was added respectively. 24 hours after infection, the medium was replaced with 5% FBS and cultured for another 48 hours to induce the production and release of P1 virus-like particles into the supernatant. Cells were collected, and intracellular luciferase activity was detected using a dual-luciferase assay kit. Luciferase activity was also detected using a multi-mode microplate reader to quantify the replication level of P1 virus-like particles in the two groups.

[0041] The results are as follows Figure 3 As shown, after P1 target cells were infected with P0 virus-like particles derived from EGLN1-KO cells, the replication level of P1 virus-like particles was significantly higher than that of wild-type cell-derived groups.

[0042] (2.3) Inhibition of P0 / P1 virus-like particles by EGLN1 overexpression Following the experimental procedures for detecting P0 and P1 virus-like particles in sections 2.1 and 2.2 above, EGLN1-flag plasmid was additionally co-transfected into HEK293T wild-type cells, and an empty vector transfection group was set up as a control group. Both EGLN1-flag plasmid and empty vector were transfected at 2.0 μg.

[0043] Subsequent transfection, culture, and detection steps were the same as those in Sections 2.1 and 2.2 for the EGLN1 knockout group. The differences in luciferase activity of P0 and P1 virus-like particles and VP40 gene copy number between the overexpression group and the control group were compared.

[0044] The results are as follows Figure 4 , Figure 5 As shown, after EGLN1-Flag overexpression, the replication level of P0 virus-like particles and the expression level of the viral genome VP40 gene in the supernatant were significantly lower than those in the empty vector control group; the difference was statistically significant. Figure 4 ); EGLN1-Flag overexpression significantly inhibited P1 virus-like particle replication and the expression level of the viral genome VP40 gene in the supernatant, with statistically significant differences. Figure 5 ).

[0045] (2.4) Detection of the promoting effect of EGLN1 inhibitor on P0 virus-like particles On day 1, HEK293T cells were seeded in 6-well plates at 4 × 10⁶ cells per well.5 One cell was cultured at 37°C and 5% CO2 for 24 hours to produce P0 cells.

[0046] On the second day, the trVLP system plasmid and the PGL4-Basic internal reference plasmid were transfected according to the same plasmid ratio as described in Section 2.1 to complete the transfection operation.

[0047] Twenty-four hours after transfection, the culture medium was replaced with one containing 5% FBS, and three inhibitors at different concentration gradients were added: DMOG (50 μM, 100 μM, 200 μM), CoCl2 (25 μM, 50 μM, 100 μM), and IXO4 (10 μM, 50 μM, 250 μM). A blank control group without EGLN1 inhibitor was also included. Twenty-four hours after EGLN1 inhibitor treatment, cells were collected to detect dual-luciferase activity and quantify the replication level of P0 virus-like particles.

[0048] The results are as follows Figure 6 As shown, after treatment with EGLN1 inhibitors DMOG, CoCl2, and IXO4 in a concentration gradient, dual-luciferase assays showed that the replication level of P0 virus-like particles increased with increasing EGLN1 inhibitor concentration.

[0049] 3. Detection of the inhibitory effect of HIF1α interference (siHIF1α) on P0 virus-like particles in the Ebola virus trVLP system. (3.1) siRNA transfection and HIF1α knockdown verification HEK293T cells were seeded in 6-well plates at a density of 4 × 10⁶ cells per well. 5 Each cell was cultured for 24 hours until the cell confluence reached 60-70%.

[0050] Using Lipfectamin 3000, 2 μg of HIF1α siRNA and negative control siRNA (NC group) were transfected into cells. After culturing for 48 hours, cell lysates were collected, and HIF1α protein levels were detected by Western blot to verify the knockdown efficiency.

[0051] The results are as follows Figure 7 As shown, Western blot analysis revealed that the expression level of HIF1α protein in the siHIF1α group was significantly reduced, with a knockdown efficiency of >80%.

[0052] (3.2) Detection of the inhibitory effect of HIF1α on P0 virus-like particles For cells that were successfully knocked down, transfected with the trVLP system plasmid and the PGL4-Basic internal control plasmid according to the same plasmid ratio as in Section 2.1, as P0 generating cells.

[0053] After transfection, cells were cultured under the conditions described in Section 2.1. Cells were collected to detect dual-luciferase activity and quantify the replication level of P0 virus-like particles.

[0054] The results are as follows Figure 8 As shown, in cells where HIF1α interference was successful, the replication level of P0 virus-like particles was significantly lower than that in the NC group, as detected by dual-luciferase assay.

[0055] 4. Detection of the inhibitory effect of HIF1α inhibitors on the replication of P0 and P1 virus-like particles in the Ebola virus trVLP system. (4.1) Inhibition experiment of HIF1α inhibitor on P0 virus-like particles On day 1, HEK293T cells were seeded in 6-well plates at 4 × 10⁶ cells per well. 5 One cell was cultured for 24 hours to produce P0 cells.

[0056] On day 2, P0-producing cells were transfected with the trVLP system plasmid and the PGL4-Basic internal control plasmid according to the same plasmid ratio as described in section 2.1. The medium was replaced with one containing 5% FBS 24 hours after transfection.

[0057] 10 μM BAY 87-2243 and 50 μM PX-478 2HCl were added, respectively, to form a control group without HIF1α inhibitors, and the cells were cultured for 24 hours. Cells were collected, and dual-luciferase activity was measured to quantify the replication level of P0 virus-like particles.

[0058] The results are as follows Figure 9 As shown, treatment with the HIF1α inhibitors BAY 87-2243 and PX-478 2HCl reduced the replication level of P0 virus-like particles, which significantly inhibited the replication of P0 virus-like particles in the Ebola virus trVLP system.

[0059] (4.2) Inhibition experiment of HIF1α inhibitor on P1 virus-like particles Following the preparation procedure for P1 virus-like particles in Section 2.2, P0 virus-like particles generated after treatment with the aforementioned HIF1α inhibitor were used to infect new HEK293T target cells. At the time of infection, corresponding concentrations of the two inhibitors were added simultaneously. Twenty-four hours after infection, the medium was replaced with one containing 5% FBS, and the cells were cultured for another 48 hours. Cells were then collected to detect dual-luciferase activity.

[0060] The results are as follows Figure 10 As shown, treatment with the HIF1α inhibitors BAY 87-2243 and PX-478 2HCl reduced the replication level of P1 virus-like particles, confirming the inhibitory effect of HIF1α inhibitors on the replication of P1 virus-like particles.

[0061] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of substances targeting the EGLN1-HIF1α signaling axis in the preparation of drugs for treating Ebola virus infection.

2. The application according to claim 1, characterized in that, The substance targeting the EGLN1-HIF1α signal axis has any one of the following functions (1)-(2): (1) Promotes the expression level of the EGLN1 gene; (2) Interfere with or reduce the expression level of the HIF1α gene.

3. The application according to claim 1, characterized in that, The substance targeting the EGLN1-HIF1α signaling axis promotes the expression level of the EGLN1 gene, thereby inhibiting intracellular Ebola virus replication and suppressing the expression level of the Ebola virus genome VP40 gene.

4. The application according to claim 1, characterized in that, The substance targeting the EGLN1-HIF1α signal axis includes any one of the following (1)-(5): (1) EGLN1 agonists; (2) Overexpression plasmid of EGLN1 gene; (3) HIF1α inhibitors; (4) HIF1α siRNA; (5) Gene editing tools that knock out HIF1α gene expression.

5. The application according to claim 4, characterized in that, The overexpression plasmid of the EGLN1 gene was constructed by inserting the CDS sequence of the EGLN1 gene into the pcdna3.1(+) plasmid.

6. The application according to claim 4, characterized in that, The HIF1α inhibitor is either BAY 87-2243 or PX-4782HCl.

7. The application according to claim 6, characterized in that, The HIF1α siRNA is an siRNA formed by annealing two single-stranded molecules as shown in SEQ ID No. 1 and SEQ ID No. 2: SEQ ID No.1: 5'-CAACCUCAGUGUGGGUAUATT-3'; SEQ ID No. 2: 5'-UAUACCCACACUGAGGUUGTT-3'.

8. A drug for treating Ebola virus infection, characterized in that, The drug is used to interfere with or reduce the expression level of the HIF1α gene or promote the expression level of the EGLN1 gene.

9. A drug for treating Ebola virus infection according to claim 8, characterized in that, The drug includes any one of the following (1)-(5): (1) EGLN1 agonists; (2) Overexpression plasmid of EGLN1 gene; (3) HIF1α inhibitors; (4) HIF1α siRNA; (5) Gene editing tools that knock out HIF1α gene expression.

10. A method for designing a drug for treating Ebola virus infection, for non-disease treatment and diagnosis purposes, characterized in that, Targeting the EGLN1-HIF1α signaling axis, the expression of the HIF1α gene is interfered with or reduced, or the expression of the EGLN1 gene is promoted.

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