An inhibitor that inhibits viral replication and cancer cell proliferation

Abstract

The present invention relates to the field of biomedicine. The present invention provides the application of mammalian MARCH proteins in anti-virus and anti-tumor, wherein human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins and mouse MARCH2 proteins can significantly block the cleavage and transport of viral membrane proteins, inhibit the activity of Furin protease, and block the replication of cancer cells; it provides valuable reference for subsequent anti-virus and anti-cancer research, and may promote the research and development of new anti-enveloped virus infection and cancer treatment strategies.

Description

Technical Field

[0001] The present invention relates to the field of virus infection and tumor cell proliferation, and specifically to an inhibitor related to MARCH protein. Background Art

[0002] The process of enveloped virus infection can be divided into three steps: invasion and membrane fusion, transcription and replication, assembly and release; invasion and membrane fusion are the first step of viral infection and are also important targets for antiviral intervention. Therefore, blocking the invasion and membrane fusion pathways of important enveloped viruses such as influenza, Middle East respiratory syndrome, and Ebola can achieve the goal of inhibiting viral infection and treating viral diseases.

[0003] At present, some host cell membrane-localized proteins have been found to have antiviral functions. Among them, MARCH protein was found to be homologous to the E3 ligases of K3 and K5 of Kaposi's sarcoma-associated herpes virus (KSHV). As an innate immune antiviral factor, it can exert its antiviral activity at different stages of viral replication. Since MARCH protein has the function of immune recognition, it is also called immune recognition protein (MIRs).

[0004] The membrane-associated RING-CH (MARCH) family is a family of RING finger domains belonging to E3 ubiquitin ligases discovered in recent years. So far, 11 MARCH family members have been discovered, namely MARCH1 to 11, most of which have similar molecular structures, including a cytoplasmic N-terminal C4HC3RING domain and two or more transmembrane domains, but MARCH7 and MARCH10 do not contain predictable transmembrane domains. Members of the MARCH family are widely distributed and participate in a variety of cellular functions, such as immune regulation, protein quality control and membrane transport, endoplasmic reticulum-related degradation, endosomal protein transport, mitochondrial dynamics balance and spermatogenesis regulation. Studies have shown that MARCH proteins inhibit viral replication using two antiviral modes; on the one hand, MARCH proteins downregulate the cell surface expression level of viral envelope glycoproteins through E3 ubiquitin ligase activity and induce their intracellular degradation. This mechanism has viral intracellular sequence dependence (cytoplasmic tail-dependence, CTD), including VSG, MLV, NiV envelope glycoproteins and IAV M2 proteins; on the other hand, MARCH proteins retain viral envelope glycoproteins in cells but do not induce their degradation. They have viral envelope glycoprotein intracellular sequence independence (cytoplasmic tail independence (CTI), including HIV-1, EBOV and SARS-CoV-2 envelope glycoproteins. It has been reported that human MARCH1 and MARCH2 can effectively inhibit the infection activity of HIV-1, and MARCH8 can inhibit the maturation of EBOV GP. Tada et al first reported that MARCH8 protein can inhibit HIV-1 and VSV replication by targeting viral envelope glycoproteins. Patent CN 111781377 MARCH8 in B inhibits the N-glycosylation of class I fusion proteins and blocks the shedding and secretion of class I fusion proteins, thereby hindering their maturation, resulting in a decrease in the expression of class I fusion proteins in host cells, thereby providing a basis for identifying the status of host cell fusion viruses and screening methods for antiviral active drugs.

[0005] Furin, as a member of the proprotein convertase (PC) family, is responsible for cleaving host cell precursor proteins. The full length of furin includes an N-terminal signal peptide, a propeptide, a catalytic domain, a conserved P domain, a Cys-rich domain, a transmembrane domain, and a cytoplasmic domain. Among them, the N-terminal signal peptide can provide a furin transport signal, further guiding the newly synthesized furin precursor into the endoplasmic reticulum-Golgi secretion pathway; the propeptide plays a vital role in the folding, activation, and transport of PC. The sequence homology of the catalytic domain of furin and other PCs is 54-70%, and the aspartic acid (Asp), histidine (His), and serine (Ser) residues that form the catalytic triad are strictly conserved. This is critical for enzyme activity, pH regulation, and calcium requirements. Furin is anchored to the membrane structure in the cell through the transmembrane region, while the cytoplasmic domain is responsible for controlling the localization and classification of Furin in the trans-Golgi network (TGN) / endosome system. Furin can cleave precursor proteins and is essential for maintaining the stability of the intracellular environment. In addition, Furin is also used by many viruses and bacteria. After the precursor proteins of certain diseases are cleaved, they participate in the regulation of many key biological processes in the cell; they can mediate the proteolytic cleavage of multiple hosts and pathogens and promote the maturation of viral envelope glycoproteins; at the same time, Furin or other PC cleavage of viral envelope glycoproteins plays an important role in viral infection of target cells. Most viruses need their glycoproteins to be hydrolyzed before they can infect host cells. In some cases, the cleavage of glycoproteins is a prerequisite for viral pathogenicity, such as playing an important biological role in the infection process of coronaviruses, flaviviruses, human immunodeficiency virus, Marburg virus and Ebola virus. Summary of the invention

[0006] Current studies have shown that MARCH proteins can inhibit the envelope glycoprotein activity of EBOV, new coronavirus (SARS-CoV-2), murine leukemia virus (MLV), influenza virus (IAV) and Nipah virus (NiV), as well as the M2 protein activity of IAV. Mainstream research focuses on the retention of HIV-1Env by human MARCH8 in the TGN and the blocking of the cleavage and glycosylation maturation of EBOV GP envelope glycoprotein and SARS-CoV-2S envelope glycoprotein. At the same time, MARCH8 can also downregulate MHC II molecules on the cell surface and limit the replication of HIV-1. Based on the in-depth research results of MARCH8 in the MARCH family, the other members of MARCH (MARCH1 / 2), bovine MARCH protein (MARCH1 / 2) and mouse MARCH protein (MARCH1 / 2) were further studied. The results showed that human MARCH1 and MARCH2, bovine MARCH2 and mouse MARCH1 can effectively inhibit EBOV GP cleavage, that is, mammalian MARCH proteins are conservative in blocking viral replication in an independent (CTI) mode through the intracellular sequence of viral envelope glycoproteins. The sequences of MARCH1 and MARCH8 are highly homologous, but not with MARCH2, but they can both block the intracellular cleavage of EBOV GP protein.

[0007] Our further research shows that MARCH molecules enter the Golgi apparatus through the endoplasmic reticulum, and are transported from the endosome to the plasma membrane (PM) surface, where they regulate the expression of the molecule-dependent E3 ubiquitin ligase activity on the cell membrane surface; after the initial N-glycosylation in the endoplasmic reticulum, the EBOV GP envelope glycoprotein molecule enters the Golgi apparatus for complex N-glycosylation and O-glycosylation, and is then cleaved by the host cell furin protease (Furin) and forms a trimer of its own heterodimer, which is transported from the endosome to the plasma membrane (PM) to package the viral genome when the virus is released. When MARCH1 / 2 / 8 molecules are present, the activity of Furin in cleaving EBOV GP in the Golgi apparatus is blocked; at the same time, MARCH1 / 2 / 8 molecules retain the Furin / EBOVGP complex in the Golgi apparatus, blocking the further transport of EBOV GP to the cell membrane surface, thereby blocking the virus replication and infection cycle, thereby exerting its antiviral activity;

[0008] The present invention was completed based on further studies of this mechanism.

[0009] In a first aspect, the present invention provides an antiviral drug, comprising a MARCH protein or a nucleic acid encoding the MARCH protein, wherein the MARCH protein is selected from one or more of human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins, and mouse MARCH2 protein, wherein the MARCH protein or the nucleic acid encoding the MARCH protein is derived from body cells or is artificially synthesized, and the drug can significantly block the cleavage and transport of viral membrane proteins.

[0010] Furthermore, the virus is preferably an envelope protein virus, including influenza virus, Middle East respiratory syndrome coronavirus, Ebola virus, African swine fever virus, blue ear disease virus, pseudorabies virus, epidemic diarrhea virus, Newcastle disease virus, human respiratory syncytial virus, measles virus, mumps virus, and newly emerging zoonotic viruses such as Menengao virus, Hendra virus, and Nipah virus.

[0011] Furthermore, the antiviral drug contains a pharmaceutically acceptable carrier.

[0012] Furthermore, the antiviral drug also contains another antiviral drug different from the MARCH protein or a second active drug that enhances the body's immunity, and the second active drug is selected from iodine, amantadine, ribavirin sodium foscarnet, acyclovir, interferon α, interferon α-2a, interferon 2b, zanamivir, oseltamivir, sofosbuvir, telaprevir, boceprevir, enfuvirid, zidovudine, tenofovir disoproxil, didanosine, abacavir, emtricitabine, zalcitabine, lamivudine, stavudine, etc.

[0013] In a second aspect, the present invention provides an anti-cancer drug, comprising a MARCH protein or a nucleic acid encoding the MARCH protein, wherein the MARCH protein is selected from one or more of human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins, and mouse MARCH2 protein, and the MARCH protein or the nucleic acid encoding the MARCH protein is derived from body cells or is artificially synthesized, and the drug can significantly inhibit the activity of Furin protease and block the replication of cancer cells.

[0014] Furthermore, the cancer includes lung cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer, etc.

[0015] In a third aspect, the present invention provides a use of a MARCH protein or a nucleic acid encoding the MARCH protein in preparing a drug for treating a disease.

[0016] Furthermore, the diseases include viral infectious diseases and cancers. The viral infectious diseases include influenza, Middle East respiratory syndrome, Ebola virus disease, African swine fever, blue ear disease, pseudorabies, epidemic diarrhea and other diseases; the cancers include lung cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, gastric cancer, cervical cancer, ovarian cancer, breast cancer and the like.

[0017] In a fourth aspect, the use of a MARCH protein or a nucleic acid encoding the MARCH protein in the preparation of a Furin inhibitor, wherein the MARCH protein is selected from one or more of human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins, and mouse MARCH2 protein, and the MARCH protein or the nucleic acid encoding the MARCH protein is derived from body cells or is artificially synthesized.

[0018] In a fifth aspect, the use of a MARCH protein or a nucleic acid encoding the MARCH protein in the preparation of a kit for detecting viral infection, wherein the MARCH protein is selected from one or more of human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins, and mouse MARCH2 protein, and the MARCH protein or the nucleic acid encoding the MARCH protein is derived from body cells or is artificially synthesized.

[0019] The kit comprises a reagent capable of detecting MARCH protein or a nucleic acid encoding the MARCH protein. When the detected MARCH protein or the nucleic acid encoding the MARCH protein is highly expressed, it indicates that the risk of the host cell being infected by the virus is significantly increased.

[0020] In a sixth aspect, the present invention provides the use of a MARCH protein or a nucleic acid encoding the MARCH protein in screening antiviral drugs, wherein the MARCH protein is selected from one or more of human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins, and mouse MARCH2 protein. The use means that when the expression level of the MARCH protein or the nucleic acid encoding the MARCH protein in the host cell increases, the drug does not have the effect of controlling viral infection, and when the expression level of the MARCH protein or the nucleic acid encoding the MARCH protein in the host cell decreases, the drug has the effect of controlling viral infection, and the drug can be considered for development as an antiviral drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 MARCH proteins block cleavage of EBOV GP

[0022] A. EBOV GP (full length) was co-expressed in 293T cells with human / bovine / mouse MARCH1 / 2 or human MARCH 8. GP was detected with FLAG antibody, and HA-tagged MARCH protein was detected with HA antibody.

[0023] B. HeLa cells were transfected with human / bovine / mouse MARCH1 / 2-GFP vectors, cell nuclei were stained with DAPI, and the fluorescence signals were detected using a confocal microscope.

[0024] C. GPΔMLD, pNL-Luc-ΔEnv and MARCH molecule expression vectors were co-transfected into 293T cells. The supernatant containing pseudovirions was collected to infect Vero cells. The infectivity was expressed by luciferase value.

[0025] D. VSV-G, pNL-Luc-ΔEnv and MARCH1 / 2 molecule expression vectors were co-transfected into 293T cells. The supernatant containing pseudovirions was collected to infect Vero cells. The infectivity was expressed by luciferase value.

[0026] E. EBOV GP and MARCH1 / 2 molecule expression vectors were co-transfected into Huh7 cells. Samples were harvested 36 hours later for WB detection. Anti-VSV-G was used to detect VSV-G, and HA antibody was used to detect HA-tagged MARCH1 / 2. M: MARCH protein; HM: human MARCH protein; BM: bovine MARCH protein; M: mouse MARCH protein.

[0027] Figure 2 Intracellular co-localization analysis of MARCH protein and EBOV GP

[0028] A. GPΔMLD-GFP was co-expressed with MARCH1 / 2 / 8 in HeLa cells. MARCH1 / 2 / 8 was detected by HA antibody and cell nuclei were detected by DAPI. Fluorescence signals were monitored by confocal microscopy.

[0029] B. MARCH-VN / GPΔMLD-VC BiFC pair was co-expressed with CNX-mCherry or TGN46-mCherry in HeLa cells, and the nuclei were stained with DAPI. Fluorescence signals were collected by confocal microscopy. M1: MARCH1; M2: MARCH2; M8: MARCH8.

[0030] Figure 3 MARCH protein inhibits intracellular cleavage of EBOV GP by binding to Furin

[0031] A. MARCH-VN / furin-VC BiFC pair was co-transfected into 293T cells. Intracellular fluorescence signal was detected by flow cytometry. B. MARCH-VN / furin-VC BiFC pair was co-transfected into HeLa cells. MARCH-VN protein contains a C-terminal HA fusion tag. Cells were stained with fluorescent HA antibody to detect MARCH protein and DAPI to detect cell nuclei. Fluorescence signal was obtained under confocal microscope.

[0032] C. Detection of intracellular co-expression of EBOV GP, Furin and MARCH1 / 2 / 8. FLAG antibody detects GP and Furin, and HA antibody detects MARCH1 / 2 / 8. SP: signal peptide.

[0033] Figure 4 Identification of the key domains of Furin's interaction with human MARCH1 / 2 / 8. Full-length and ΔSD / ΔP mutants of Flag-tagged Furin were co-immunoprecipitated with HA-tagged MARCH1 / 2 / 8. FLAG antibody detected Furin, and HA antibody detected MARCH1 / 2 / 8. ΔSD / ΔP: Furin ΔSD / ΔP mutant.

[0034] Figure 5 Diagram of the intracellular working mode of MARCH1 / 2 / 8 molecules DETAILED DESCRIPTION

[0035] The envelope of enveloped viruses is a lipid bilayer. The protein molecules on the envelope are encoded by the viral genome and often exist in the form of glycoproteins or lipoproteins. They can mediate the fusion of the viral envelope with the host cell membrane or endosome membrane, thereby releasing the viral contents into the host cells and infecting the host cells.

[0036] Ebola virus (EBOV) is a highly virulent pathogen that causes Ebola virus disease (EVD), resulting in high mortality in humans. The EBOV genome encodes several structural proteins, namely nucleoprotein (NP), viral protein 35 (VP35), VP40 and envelope glycoprotein (GP), viral protein VP30, viral protein VP24 and transcription polymerase (L). Due to the RNA transcription sliding editing mechanism, EBOV GP gene transcription produces three transcripts, encoding GP, soluble GP (sGP) and small soluble GP (ssGP), respectively. Membrane-localized GP is a type I membrane protein, accounting for approximately 25% of the total GP product, and is essential for viral infection of target cells. The EBOV GP0 precursor undergoes initial N-glycosylation modification in the endoplasmic reticulum (ER) and is then transported to the Golgi apparatus to complete its mature N-glycosylation and O-glycosylation. Mature N, O-glycosylated GP0 is cleaved into two subunits (GP1 and GP2) by furin at a highly conserved site (RXA / RR). GP1 and GP2 subunits are reconnected by disulfide bonds to form GP1,2, which are then assembled into trimers and transported to the cell membrane surface. GP1 contains a mucin-like domain (MLD), which is N-glycosylated and highly O-glycosylated at the same time, significantly increasing its molecular weight. Therefore, in Western blot (WB) assays, the molecular weight of mature glycosylated GP1 is usually larger than that of full-length immature GP0.

[0037] Furin, as a member of the PC family, can cleave substrate proteins, namely precursor proteins, which are necessary for the growth and replication of various cells. After the precursor proteins are cleaved, they participate in the regulation of many key biological processes in the cell. Furin can also recognize many viral envelope glycoproteins, including HIV-1, EBOV, IAV H5N1 and SARS-CoV-2. Cutting viral envelope glycoproteins by Furin or other PCs is important for viral infection of target cells. In this study, we verified that human MARCH1 and MARCH2 have intracellular interactions with Furin, thereby blocking Furin's cleavage of EBOV GP, and further proved that MARCH1 and MARCH2 retained the MARCH1 / MARCH2-furin-EBOV GP complex in the TGN, while blocking the host Furin activity and GP-mediated viral infection ability; in addition, studies have shown that MARCH1 / 2 / 8 can specifically bind to Furin, block its protease activity, and inhibit its substrate cleavage activity; when cancer cells grow and multiply, the expression level of the required cell replication proteins is high, and all require Furin cleavage to complete their protein activity, that is, furin activity is positively correlated with the growth rate of cancer cells; therefore, the MARCH protein in the present invention can also inhibit Furin's cleavage of proteins necessary to maintain rapid replication when expressed in cancer cells, thereby inhibiting the growth of cancer cells, so the MARCH protein expression or purification in the present invention has anti-cancer efficacy.

[0038] The "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers that are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. In the composition, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, or a buffer. In addition, auxiliary substances may also be present in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc.

[0039] Example 1 Experimental method

[0040] 1.1 Materials and methods

[0041] (1) Cell lines: human embryonic kidney 293T cells, cervical cancer HeLa cells, African green monkey kidney Vero cells, and human Huh7 cells were cultured at 37°C and 5% CO2 in DMEM (Gibco) supplemented with 8% fetal bovine serum (FBS) (Gibco), streptomycin, and penicillin (100 μg / mL). All cell lines were obtained from the American Type Culture Collection (ATCC) and stored in our laboratory.

[0042] (2) DNA Construction Bovine MARCH1 and MARCH2 genes were obtained from bovine peripheral blood mononuclear cells (PBMCs) by RT-PCR amplification and cloned into the pCAGGS vector with a C-terminal HA tag through the EcoRI / XhoI restriction sites. Human MARCH family members MARCH1 and MARCH2 were amplified from Origene cDNA clones RC207517 (GenBank#NM_001166373) and RC229839 (GenBank#NM_016496), respectively, and then cloned into the pCAGGS vector with a C-terminal HA tag through the EcoRI / XhoI restriction sites. Mouse MARCH1 (GenBank#NM_001166372) and MARCH2 (GenBank#NM_145486) were cloned into the pCAGGS vector with a C-terminal HA tag through the ClaI / XhoI restriction sites (Co-Mei, Changchun, China). The MARCH1 or MARCH2 gene was inserted into the pEGFP-N1 vector (GenBank #U55762) through the EcoRI / AgeI restriction sites to construct human / bovine / mouse MARCH1-GFP and MARCH2-GFP expression vectors. Human MARCH1 / MARCH2 was cloned into the pcDNA3.1-VN vector through the EcoRI / AgeI restriction sites to become pcDNA3.1-MARCH1 / MARCH2-VN. The human NHA-MARCH8 expression vector, N-terminal FLAG-tagged EBOV GPΔMLD expression vector, full-length EBOV GP expression vector, Furin / Furin-ΔSD (Furin SD deletion) expression vector, Furin-ΔP (Furin P deletion) expression vector, human MARCH8-GFP / EBOV GP-ΔMLD-GFP expression vector, Furin-VC / hMARCH8-VN / GP-ΔMLD-VN expression vector, CRT-mCherry / TGN46-mCherry expression vector, and pNL-Luc-ΔEnv reporter proviral vector were constructed and maintained by our laboratory.

[0043] (3) Confocal microscopy was used to detect the expression of HeLa cells (2 × 10 5) and transfected with Lipofectamine 3000 reagent (Thermo Fisher). Twenty-four hours later, the cells were washed, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. After washing, the cells were blocked with 5% bovine serum albumin (BSA). For immunofluorescence detection, the cells were incubated with anti-HA primary antibody for 1 hour at room temperature. After washing three times, the cells were stained with Alexa Fluor's 647-conjugated secondary antibody for 1 hour at room temperature. After washing three times with PBS, the cell nuclei were stained with DAPI for 30 minutes, washed at room temperature for 1 hour and used for confocal microscopy (Zeiss LSM800). At least 100 random fields of view were analyzed for each dish, and the most representative images were selected for display.

[0044] (4) Flow cytometry analysis 293T cells (2×10 5 ) were seeded in 6-well plates, and Furin-VC and MARCH1 / 2-VN expression vectors were co-transfected into 293T cells. 48 hours after transfection, the cells were centrifuged at 2000 rpm for 5 minutes and fixed with 4% paraformaldehyde for 10 minutes at room temperature. After collection by centrifugation, flow cytometry analysis was performed.

[0045] (5) Immunoblotting assay: 293T cells (5×10 5 or 2×10 6 ) were seeded in 6-well plates or 10 cm culture dishes. 36 hours after transfection, cells were lysed with RIPA buffer (Sigma) and the supernatant was obtained by centrifugation, gently mixed with the loading dye, and boiled in a water bath for 5 minutes. Subsequently, the samples were placed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane and blocked with 4% skim milk for 1 hour at room temperature. After washing, the PVDF membrane was incubated with the primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody. Alternatively, the PVDF membrane was incubated directly with the HRP-conjugated primary antibody. Finally, the PVDF membrane was exposed to enhanced chemiluminescence (ECL).

[0046] (6) Antibodies: Mouse anti-actin, mouse anti-HA, and mouse anti-FLAG monoclonal antibodies were purchased from Sigma; HRP-conjugated anti-mouse immunoglobulin G secondary antibody was purchased from Pierce; Alexa Fluor 647-conjugated secondary antibody was purchased from Thermofisher; HRP-conjugated anti-HA and anti-FLAG primary antibodies were purchased from Roche and Sigma, respectively; ECL detection reagent was purchased from Thermofisher.

[0047] (7) Immunoprecipitation experiment: 2×10 6293T cells were seeded in 10 cm culture dishes and transfected 12 h later. 48 h after transfection, cells were lysed with RIPA buffer and the supernatant was collected by centrifugation. Magnetic beads coupled with anti-FLAG antibody were incubated overnight at 4 °C. The beads were then collected with a magnetic stand, washed repeatedly and used for WB detection.

[0048] (8) Virus infectivity test: 12 hours before transfection, use 5×10 5 293T cells were seeded in 6-well plates, and 293T cells were co-transfected with pcDNA3.1-FLAG-GP-ΔMLD, pNL-Luc-ΔEnv and MARCH protein expression vectors. 48 hours after transfection, the supernatant containing viral particles was collected. After quantitative detection, the indicator Vero cells were infected. After 48 hours, the Vero cells were lysed and the virus infectivity was determined by luciferase activity assay.

[0049] (9) Luciferase activity detection One day before virus inoculation, 1×10 4 Vero cells were inoculated in 96-well plates, and then the supernatant containing viral particles was added to the Vero cells. After 48 hours, the cell culture was terminated. The cells were washed 3 times with PBS and lysed with 100 μL RIPA buffer. 100 μL of cell lysate was taken and mixed with an equal amount of substrate (Bright-Glo luciferase quantification system, Promega) at room temperature for 10 minutes and used for luminescence activity measurement.

[0050] Example 2 Analysis of the effect of human and other mammalian MARCH proteins on full-length EBOV GP

[0051] 2.1 Cloned bovine and mouse MARCH1 and MARCH2 and tracked their intracellular distribution.

[0052] 2.2 Green fluorescent protein (GFP) tags were inserted into the C-termini of MARCH1 and MARCH2 to form MARCH1-GFP and MARCH2-GFP.

[0053] 2.3 Detection of intracellular cleavage of EBOV GP by human, bovine and mouse MARCH1 and MARCH2 proteins

[0054] Human MARCH1 and MARCH2, bovine MARCH2, and mouse MARCH1 can block the cleavage of EBOV GP, while mouse MARCH2 almost loses this inhibitory effect; in the presence of bovine MARCH1, the expression of EBOV GP0 and GP1GP are both blocked, indicating that the mode of inhibition of viral envelope by other MARCH proteins is different (see Figure 1 A).

[0055] 2.4 Proposed detection of MARCH1 and MARCH2 cleavage of full-length EBOV GP and other mammalian MARCH1 and MARCH2 cleavage of EBOV GP

[0056] MARCH proteins showed membrane localization and intracellular scattered distribution, which is consistent with the intracellular localization characteristics of MARCH8 (see Figure 1 B).

[0057] 2.5 Detection of MARCH protein blocking viral replication in vitro

[0058] The EBOV GP-ΔMLD-mediated pseudovirus infection system was used to detect its function. Compared with the control group, the expression of other MARCH proteins except mouse MARCH2 could significantly reduce viral infection, which is consistent with the role of these MARCH proteins in blocking EBOV GP cleavage (see Figure 1 C). Therefore, these results indicate that mammalian MARCH1 and MARCH2 have a blocking effect on the cleavage of EBOV GP protein. It is noteworthy that human MARCH1 and MARCH2 can also significantly block the pseudotype virus infection activity mediated by VSV-G ( Figure 1 D); and, human MARCH1 and MARCH2 can inhibit the cleavage of EBOV GP in human hepatocytes ( Figure 1 E).

[0059] Example 3 Detection of MARCH protein inhibiting the expression of EBOV GP1 on the cell surface

[0060] Human MARCH1 and MARCH2 retain EBOV GP in the TGN, and mature EBOV GP1 is present on the cell surface and integrated into budding virions. Human MARCH1 and MARCH2 reduce the expression level of EBOV GP1 and reduce GP1,2-mediated viral infectivity. Therefore, it is necessary to detect whether MARCH1 and MARCH2 inhibit the expression of EBOV GP1 on the cell surface.

[0061] 3.1 EBOV GP-ΔMLD-GFP was co-transfected with human MARCH1 and MARCH2.

[0062] 3.2EBOV GP-ΔMLD-GFP is localized on the cell membrane surface, while human MARCH1 and MARCH1 proteins show membrane localization and intracellular scattered distribution characteristics (see Figure 2 A). In the presence of human MARCH1 or MARCH2, EBOV GP-ΔMLD-GFP protein was significantly downregulated from the cell membrane and retained inside the cell ( Figure 2 A).

[0063] These results suggest that human MARCH1 and MARCH2 retain EBOV GP-ΔMLD-GFP in intracellular organelles, preventing it from being further transported to the cell membrane surface.

[0064] 3.3 The above results show that human MARCH1 and MARCH2 retain EBOV GP-ΔMLD-GFP in intracellular organelles. In order to further detect the specific organelles in the cell, the following experiments were performed:

[0065] (1) The bimolecular fluorescence complementation (BiFC) system was introduced. The C-terminus of the MARCH protein was fused to the N-terminus of the green fluorescent protein Venus (VN, amino acids 2-173), while the EBOVG-ΔMLDC terminal was fused to the C-terminus of Venus (VC, amino acids 154-238).

[0066] (2) Calnexin-mCherry (CNX-mCherry) and TGN46-mCherry (TGN-mCherry) were used as ER and trans-Golgi localization markers. MARCH1-VN / MARCH2-VN / MARCH8-VN and GP-ΔMLD-VC were co-transfected with CNX-mCherry or TGN-mCherry to track their intracellular localization.

[0067] (3) The results showed that there was no fluorescence signal in the single MARCH1-VN / MARCH2-VN / MARCH8-VN / GP-ΔMLD-VC transfection (data not shown). The MARCH1-VN / MARCH2-VN / MARCH8-VN and GP-ΔMLD-VC pair had little overlap with CNX-mCherry, but had strong colocalization with TGN-mCherry (see Figure 2 B) Therefore, it can be inferred that human MARCH1 and MARCH2 retain EBOV GP in the trans-Golgi tubular network (TGN).

[0068] Example 4 Human MARCH1 and MARCH2 inhibit Furin-mediated cleavage of EBOV GP

[0069] Furin is a self-cleaving protein. Mature Furin is self-cleaved at the TGN or cell surface and then secreted out of the cell. In the presence of human MARCH protein, intracellular Furin expression is enhanced ( Figure 3), which may be because MARCH proteins retain Furin in the TGN, leading to its intracellular accumulation. Our previous work showed that MARCH8 inhibits the cleavage of EBOV GP by Furin. Therefore, we further verified whether MARCH1 and MARCH2 can block the intracellular cleavage of EBOV GP by Furin.

[0070] 4.1 Detection of the phenotype by flow cytometry and laser confocal microscopy. First, flow cytometry was used to detect the intracellular interaction between MARCH1 / MARCH2 / MARCH8 and Furin (BiFC system). MARCH1-VN / MARCH2-VN / MARCH8-VN and Furin-VC (FR-VC) were co-transfected into HeLa cells for flow cytometric analysis. MARCH1 / 2 proteins and Furin had obvious intracellular co-action signals, indicating that there was interaction between them (see Figure 3 A).

[0071] 4.2 To further verify the binding of MARCH protein to Furin, the Furin expression vector and MARCH1 / MARCH2 expression vector were co-transfected into HeLa cells. The laser confocal microscopy results showed that (see Figure 3 B), FR-VC can produce synergistic signals with MARCH1-VN / MARCH2-VN / MARCH8-VN, indicating that they form an intracellular complex.

[0072] 4.3 Based on the determination of the interaction between these proteins in cells, we continued to test whether these MARCH proteins blocked the cleavage of EBOV GP by furin. EBOV mature GP0 is cleaved into GP1 (~130kDa) and GP2 (~26kDa) by endogenous furin; when exogenous furin is expressed, EBOV mature GP0 is cleaved into three parts, namely GP1, GP1* and GP2 (see Figure 3 C). Full-length EBOV GP was co-transfected with Furin in the absence or presence of MARCH proteins. The results showed that in the presence of MARCH8, the cleavage of EBOV GP by Furin was inhibited, as very little GP1, GP1*, and GP2 were produced. In the presence of MARCH8, the expression level of intracellular Furin was enhanced in a MARCH8 expression dose-dependent manner. Similarly, in the presence of MARCH1 and MARCH2, the cleavage of EBOV GP by Furin was also blocked. Therefore, these results indicate that MARCH1, MARCH2, and MARCH8 can inhibit the cleavage of EBOV GP by Furin.

[0073] 4.4 Analysis of Furin domain recognition for human MARCH1 and MARCH2

[0074] (1) To determine the specific furin domains recognized by human MARCH1, MARCH2, and MARCH8, we used two previously constructed furin mutants, FurinSD deletion mutant (FRΔSD) and Furin P deletion mutant (FRΔP), to perform co-immunoprecipitation assays with these MARCH molecules. MARCH1, MARCH2, and MARCH8 can bind to full-length furin and FRΔSD, but rarely recognize FRΔP (see Figure 4 ), the above results indicate that MARCH1 and MARC2 interact with fruin in cells, and the SD domain and P domain are crucial for the substrate cleavage function of furin, especially the furin P domain can be recognized by these MARCH proteins.

[0075] 4.5 Human MARCH1 / 2 / 8 molecular intracellular working mode

[0076] MARCH molecules enter the Golgi apparatus through the endoplasmic reticulum, and are transported from the endosomes to the surface of the plasma membrane (PM). They regulate the expression of molecules on the surface of the cell membrane by relying on their E3 ubiquitin ligase activity. After the initial N-glycosylation in the endoplasmic reticulum, the EBOV GP envelope glycoprotein molecule enters the Golgi apparatus for complex N-glycosylation and O-glycosylation. After being cleaved by the host cell furin protease (Furin), it forms a trimer of its own heterodimers, which are transported to the PM through the endosomes and package the viral genome when the virus is released. When MARCH1 / 2 / 8 molecules are present, Furin's activity of cleaving EBOV GP in the Golgi apparatus is blocked. At the same time, MARCH1 / 2 / 8 molecules retain the Furin / EBOV GP complex in the Golgi apparatus, blocking the further transport of EBOV GP to the cell membrane surface, thereby blocking the viral replication and infection cycle, thereby exerting its antiviral activity (see Figure 5 ).

Claims

1. Use of a MARCH protein or a nucleic acid encoding the MARCH protein in the preparation of a furin inhibitor, wherein the MARCH protein is selected from one or more of human MARCH1 and MARCH2 proteins, bovine MARCH1 and MARCH2 proteins, and mouse MARCH1 protein, and the MARCH protein or the nucleic acid encoding the MARCH protein is derived from a body cell or is artificially synthesized; the furin inhibitor is used to inhibit Ebola virus.

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