Method for preventing and / or treating virus infections

Abstract

The invention discloses a virus protease, EV71 2Apro and SARS-CoV-23CLpro, which can promote the extracellular level of LRPAP1 to be increased, and the LRPAP1 can degrade IFNAR1 (interferon-related protein 1) and inhibit subsequent interferon signal transduction. In addition, the inhibition of LRPAP1 by a composition containing alpha2M, siRNA, or other LRPAP1 inhibitors can improve RNA virus and DNA virus infections. The invention not only expands the relevance between lipoprotein metabolism and host innate immune response, but also lays a foundation for the development of broad-spectrum antiviral drugs.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 500,277, filed May 5, 2023, which is incorporated herein by reference in its entirety. Reference sequence list

[0002] The submitted sequence list file, named “P2730PC00_sequence_list.xml”, was created on May 3, 2024, uses the ST.26XML file format, and has a file size of 26.2KB. It is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to at least the technical fields of viral infection, drug therapy, and immunology. This invention discloses a novel mechanism for using viruses to circumvent host cellular immunity, thereby supporting a strategy for developing broad-spectrum antiviral drugs. Background Technology

[0004] The host's innate immunity is the first line of defense against viral invasion and replication. Host cells can rapidly recognize viral components through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RIG-I-like receptors, RLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs).

[0005] For example, once the PRR recognizes a pathogen or its key components, it immediately induces infected cells and other responding cells to express and secrete type I interferons (IFNα, IFNβ) and other cytokines. The crucial role of the interferon (IFN) signaling pathway in limiting or clearing pathogen invasion is well-known. Secreted IFN binds to the heterodimeric transmembrane receptor IFNα (IFNAR) and induces its phosphorylation, subsequently activating the phosphorylation of JAK and TYK kinases, further phosphorylating signal transducers and transcription activators (STAT1 / STAT2), thereby initiating the transcription of hundreds of interferon-stimulated genes (ISGs) to combat viral infection. This interferon signaling cascade constitutes the core of the host's innate immune response.

[0006] To achieve effective infection, viruses target intracellular interferon signaling pathways through various mechanisms, thereby weakening the host cell's innate immune response. This process is crucial for the infection of brain nerve cells by viruses from different viral families (such as enterovirus EV71, flavivirus Zika virus, coronavirus SARS-CoV-2, and herpesvirus HSV-1), as the central nervous system's immune response primarily relies on local cellular innate immune mechanisms. For many years, viruses have countered host defense mechanisms by inhibiting interferon (IFN) signaling intracellularly through various mechanisms. However, the mechanism by which viruses target interferon signaling via extracellular pathways has not yet been discovered. Pioneering and groundbreaking approaches are urgently needed in this field to advance the development of highly effective antiviral therapies. Summary of the Invention

[0007] In a first aspect, the present invention provides a method for preventing and / or treating viral infections. The method comprises administering an effective dose of a low-density lipoprotein receptor-associated protein 1 (LRPAP1) inhibitor to a subject in need, targeting LRPAP1 to thereby increase the expression levels of interferon and its derivatives in the subject's body.

[0008] In one embodiment of the present invention, the interferon and its derivatives include natural / recombinant interferon, conformationally modified interferon, interferon conjugates, or interferon fusion proteins.

[0009] In another embodiment of the invention, the method further includes protecting the expression of interferon α / β receptor 1 (IFNAR1) in the subject. LRPAP1 inhibitors can block the binding of the N-terminus of secreted LRPAP1 to the extracellular domain of IFNAR1.

[0010] In another embodiment of the invention, the method further includes protecting interferon α / β receptor 1 (IFNAR1) with other reagents, said reagents including peptides or small molecules that can attenuate the effect of LRPAP1, or substances that can block the degradation of IFNAR1 induced by viruses or other pathogens.

[0011] In one embodiment of the present invention, the LRPAP1 inhibitor can downregulate the expression of VP1.

[0012] In one embodiment of the invention, the subject may also be given an additional IFNAR1 agonist. The IFNAR1 agonist may be given before, after, or simultaneously with the LRPAP1 inhibitor.

[0013] In one embodiment of the present invention, the concentration range of the IFNAR1 agonist is 0.01 to 10 mg / mL.

[0014] Examples of IFNAR1 agonists may include interferon-α, interferon-β, poly(I:C)-poly(I:C), polyuridine, Toll-like receptor 7 / 8 agonists, CpG oligodeoxynucleotides, etc.

[0015] In one embodiment of the invention, the viral infection includes diseases caused by DNA viruses or RNA viruses. The DNA viruses include herpesviruses or hepatitis viruses; the RNA viruses include microRNA viruses, coronaviruses, enteroviruses, and flaviviruses.

[0016] In one embodiment of the present invention, the virus-infected cells exhibit tolerance to conventional interferon treatment.

[0017] In one embodiment of the present invention, the LRPAP1 inhibitor may be a pharmaceutical composition comprising α2-macroglobulin, an siRNA sequence with sequence number SEQ ID NO.:1 or SEQ ID NO.:2, a lysosomal inhibitor, or a combination thereof.

[0018] In another embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant.

[0019] In one embodiment of the invention, the lysosomal inhibitor includes methylamine or baffirizine.

[0020] In one embodiment of the present invention, the effective dose is within the biosafety range and does not exceed 1.5 mg / 100 mL.

[0021] In a second aspect, the present invention provides a recombinant protein of low-density lipoprotein receptor-associated protein 1 (LRPAP1), with reference protein number NP_002328.1 and an open reading frame (ORF) sequence of 1074 bp, and an α2-macroglobulin (α2M) protein, with reference protein number NP_001334352.2 and an open reading frame (ORF) sequence of 4425 bp.

[0022] This invention reveals that low-density lipoprotein-associated protein (LRPAP1) is associated with virus-induced IFNAR1 inhibition. Viral stimulation-induced LRPAP1 secretion acts as an inhibitory ligand, binding to the extracellular domain of IFNAR1 and leading to its degradation. Essentially, this invention identifies α2-macroglobulin (α2M) as a potent inhibitor of extracellular LRPAP1, capable of protecting IFNAR1 expression and enhancing innate immunity against various viral infections. Attached Figure Description

[0023] Embodiments of the invention are described in more detail below with reference to the accompanying drawings, in which:

[0024] Figure 1 A schematic diagram illustrating the mechanism by which the virus circumvents the type I interferon signaling pathway via LRPAP1.

[0025] Figure 2A Showing the identification of 2A pro The results of the pull-down assay for the interacting proteins are presented, along with the immunoblotting results of the candidate 2A interacting protein LRPAP1. Figure 2B The results of immunoblotting and RT-qPCR analysis of LRPAP1 protein and its mRNA levels in human embryonic kidney 293T (HEK-293T) cells transfected with pcDNA4B (vector), pcDNA4B-2A, or pcDNA4B-2Am, respectively, are presented. Figure 2C This shows human rhabdomyosarcoma (RD) cells with or without recombinant 2A. pro (r2A) Immunoblotting results of lysate after 1 hour of treatment. Figures 2D to 2E The study described RD cells infected with EV71 at indicated MOIs, and cell lysates were obtained at different time points, followed by immunoblotting and RT-qPCR analysis. Figure 2F Describe the relative mRNA and protein levels of LRPAP1 in HEK-293T cells transfected with pcDNA4B (vector), pcDNA4B-3C, or -3D for 48 hours.

[0026] Figures 3A to 3B This describes the results of relative viral RNA levels and viral titers in RD cells 9 hours after infection with the indicated MOI. RD cells were transfected with the indicated plasmid (empty vector or pcDNA-LRPAP1 (LRPAP1)) or siRNA (random sequence RNA (NC) or siRNA-LRPAP1 (siLRPAP1)) for 39 hours prior to viral infection.

[0027] Figure 4 The indicated protein structures (EV71 2A, 4fvb; human rhinovirus RV-C 2A, 5x45; Coxsackievirus A16 (Cox A16) 2A, 4mg3; Middle East Respiratory Syndrome Coronavirus (MERS-CoV) 3CL, 5c3n; SARS-CoV-2 3CL, 6y2e; human coronavirus 229E 3CL, 1p9s; Zika virus NS2B-NS3, 5t1v; dengue virus type 2 NS2B-NS3, 4m9i) were compared using a non-sequence correlation algorithm. The labeled amino acids represent the corresponding residue pairs after alignment. A TM-score higher than 0.5 indicates structural similarity.

[0028] Figure 5Describe the comparison results of the superposition of the indicated proteins (EV71 2A, 4fvb; SARS-CoV-2 3CL, 6y2e; SARS-CoV-2PL, 6w9c) in the three-dimensional model.

[0029] Figure 6A The relative mRNA levels of LRPAP1 in HEK-293T cells transfected with empty vector pCMV, pCMV-SARS-CoV-2-PL, or pCMV-SARS-CoV-2-3CL for 48 hours were described. Figure 6B This study describes the relative mRNA levels of LRPAP1 in RD cells infected with HCoV-OC43 at MOI 1 and cultured for 72 hours. Results are expressed as mean ± standard deviation (error bars) from three replicates. *P≤0.05, **P≤0.01, ***P≤0.001 (unpaired t-test).

[0030] Figure 7 Describe HEK-293T cells transfected with empty vector (Vector) or pCMV-Flag-3CL (3CL).

[0031] Figure 8A A schematic diagram is provided to illustrate the effect of HCoV-OC43 infection-induced extracellular LRPAP1 on viral infection. Figure 8B The image shows the levels of viral RNA inside (left) and outside (right) cells. Figure 8C Describes the cytopathic effect (CPE) resulting from intracytoplasmic vacuolation 12 hours after treatment with a sham control or anti-LRPAP1 antibody. Scale bar: 20 μm.

[0032] Figure 9 Demonstrates cytopathic effects (CPE) characterized by cell rounding and shedding. Scale bar: 100 μm.

[0033] Figure 10A The figure shows the fold change in extracellular LRPAP1 in RD cells 9 hours after viral load infection with EV71. Figure 10B The fold change of extracellular LRPAP1 in HEK-293T cells was shown 48 hours after transfection with the indicator plasmid. Figures 10C to 10D Describe the changes in relative viral RNA levels and viral titers in RD cells infected with EV71 with or without recombinant LRPAP1 (rLRPAP1) treatment.

[0034] Figure 11A A schematic diagram illustrating the use of rLRPAP1 to induce short-term viral infection. Figure 11B Describe the viral particles inside the cell after viral titration. Figure 11CWestern blot results for LRPAP1 and VP1 are presented, with VP1 expression levels quantified using ImageJ software and normalized relative to GAPDH. Results are expressed as mean ± standard deviation (error bars) of three replicates. *P≤0.05, **P≤0.01, ***P≤0.001 (unpaired t-test).

[0035] Figure 12 The diagrams depict mice after receiving different treatments, along with the neurological state and behavioral performance of each mouse.

[0036] Figure 13 This describes the detection of viral protein (VP1) expression in intestinal tissue lysates using Western blotting.

[0037] Figure 14A Describe the survival rate of each group within 7 days; Figure 14B The number of mice exhibiting severe neurological deficits (such as circling, rolling, and hind limb paralysis) per day is described.

[0038] Figure 15 The results of immunoblotting of HEK-293T cell lysates expressing the indicated gene are described, as well as the results of density analysis of IFNAR1 levels using ImageJ and normalization to GAPDH.

[0039] Figure 16A The expression levels of IFNAR1 protein in HEK-293T cells overexpressing 2A or 2Am were described. Figure 16B Description of Figure 16A Density analysis results of IFNAR1 expression levels were obtained and normalized relative to GAPDH using ImageJ software. Figure 16C Describe the expression levels of IFNAR1 and LRPAP1 in HEK-293T cells that express only 2A or co-express both LRPAP1 and 2A. Figure 16D Description of Figure 16C Density analysis results of IFNAR1 expression levels were obtained and normalized relative to GAPDH using ImageJ software. Figure 16E Western blot results for IFNAR1, LRPAP1, and the viral structural protein VP1 are presented. The expression level of IFNAR1 was quantified using ImageJ software and normalized relative to GAPDH.

[0040] Figures 17A to 17B Describe the immunoblotting results of IFNAR1 in cell lysates under LRPAP1 downregulation conditions.

[0041] Figure 18AThe downstream response of the type I interferon signaling pathway is described by assessment of the relative mRNA levels of the indicated ISG. Figure 18B The downstream response of the type I interferon signaling pathway is described by assessment of the relative mRNA levels of the indicated ISG.

[0042] Figure 19A Western blot results for IFNAR1, LRPAP1, and the viral structural protein VP4 are presented. The expression level of VP4 was quantified using ImageJ software and normalized relative to GAPDH. Figure 19B Western blot results for IFNAR1 and LRPAP1 in HEK-293T cells after treatment with an indicated concentration of rLRPAP1 for 15 min or 1 hour are presented. IFNAR1 expression levels were quantified using ImageJ software and normalized relative to GAPDH. Results are expressed as mean ± standard deviation (error bars) from three replicates. *P≤0.05, **P≤0.01, ***P≤0.001 (unpaired t-test).

[0043] Figure 20A The immunoblotting results of cytoplasmic and membrane components of HEK-293T cells after treatment with 200 nM rLRPAP1 for 1 to 10 minutes are described. Figure 20B Immunofluorescence results of RD cells infected or uninfected with EV71 (MOI=10) for 6 hours are described. Cells were double-stained with anti-IFNAR1 and anti-LRPAP1. Scale bar is 5 μm. Figure 20C Immunofluorescence images depicting RD cells treated with EV71 (MOI=10) for 6 hours or with 200 nM rLRPAP1 for 1 hour, respectively. Cells were double-stained with anti-IFNAR1 and anti-LRPAP1. Scale bar: 5 μm.

[0044] Figure 21A A schematic diagram showing two cerebral hemispheres immediately after removal and incubated in an oxygenated cuvette containing ACSF or supplemented with 200 nM rLRPAP1 ACSF. Figure 21B Demonstrating the effects of IFNAR1, LRPAP1, and Na + / K + - Immunoblotting results of ATPase. *P≤0.05, **P≤0.01, ***P≤0.001 (unpaired t-test).

[0045] Figure 22A Describe the results of flow cytometry analysis of IFNAR1 on the surface of 4T1 cells with or without rLRPAP1 treatment. Figure 22BDescribe the relative mRNA levels of IFNAR1 in HEK-293T cells under conditions of LRPAP1 overexpression or downregulation.

[0046] Figure 23A This describes the results obtained by immunoblotting analysis after immunoprecipitation with indicator antibodies from HEK-293T cells that ectopically express LRPAP1 and IFNAR1. Figure 23B The results of immunoprecipitation from brain tissue lysates from 8-week-old C57BL / 6 mice are described. Figure 23C The results of pull-down assays between the extracellular domain (ECD) of IFNAR1 and lysates of HEK-293T cells overexpressing LRPAP1 were described and analyzed by immunoblotting. Figure 23D The highest confidence docking model between LRPAP1 and IFNAR1 generated by H-dock is shown, with a confidence score of 0.7822 for the combined model.

[0047] Figure 24A This study demonstrates the effect of the LRPAP1 peptide on IFNAR1 expression. Figure 24B This study demonstrates the effect of RAPD1P1 on the downstream response of the type I interferon signaling pathway.

[0048] Figure 25 This study demonstrates the effect of the LRPAP1 peptide on viral infection.

[0049] Figure 26 The results of microthermophoresis (MST) experiments describing the binding affinity between LRPAP1 fragments (RAPD1P1, RAPD1P2) and ECD-IFNAR1 are described.

[0050] Figure 27A Immunofluorescence images of early endosomal markers EEA1 and IFNAR1 in HEK-293T cells after treatment with or without 200 nMrLRPAP1 for 1 hour. Figure 27B (Left) Immunoblotting results of lysates of HEK-293T cells treated with an indicated concentration of rLRPAP1 for 1 hour. Increased phosphorylation levels of IFNAR1 (p-IFNAR1) and p38 (p-p38) were correlated. Figure 27B (Right) shows the density analysis results of p-IFNAR1 expression level, which was quantified using ImageJ and normalized relative to IFNAR1. Figure 27C This describes the results obtained by immunoblotting analysis after immunoprecipitation using indicator antibodies from HEK-293T cells overexpressing ubiquitin, LRPAP1, and IFNAR1.

[0051] Figure 28ADescribe the results of Western blots on HEK-293T cells expressing LRPAP1 ectopically 4 hours after treatment with or without 20 mMMA. Figure 28B Describe the results of Western blots on HEK-293T cells expressing LRPAP1 ectopically, after 4 hours of treatment with or without 1 μM baffirizine.

[0052] Figure 29 This describes the results of immunoprecipitation using indicator antibodies and analysis by Western blotting from HEK-293T cells ectopically expressing ubiquitin, LRPAP1, and IFNAR1. Cells were treated with 20 mMMA for 4 hours prior to collection.

[0053] Figures 30A to 30B The effects of rLRPAP1 and RAPD1P1 on HCoV-OC43 infection are demonstrated. Figure 30C The intracellular and extracellular EV71 RNA levels, as determined by RT-qPCR, are described. Figure 30D Describe the relative mRNA level of LRPAP1 in RD cells 24 hours after HSV-1 infection. Figure 30E Describe the cytopathic effect (CPE) of EV71, characterized by cell rounding and shedding. Scale bar is 20 μm. Figure 30F The effects of rLRPAP1 and RAPD1P1 on HSV-1 infection were demonstrated. Figure 30G The effects of rLRPAP1 and LRPAP1 peptides (RAPD1P1, RAPD1P2) on HSV-1 and HCoV-OC43 infection are shown. Scale bar is 100 μm. Figure 30H Describe the intracellular HSV-1 RNA levels as determined by RT-qPCR. Figure 30I Description via TCID 50 The viral titer of HCoV-OC43 was determined experimentally. Figure 30J The CPE of HSV-1 was described, characterized by cell rounding and shedding. The scale bar was 20 μm. Results are expressed as mean ± standard deviation (error bar) of three replicates. *P≤0.05, **P≤0.01, ***P≤0.001 (unpaired t-test).

[0054] Figure 31 Description by 3CL pro A schematic diagram illustrating the effect of transfection-induced extracellular LRPAP1 on EV-A71 and HSV-1 infection.

[0055] Figure 32The images depict the following: (left) immunoblotting results of HEK-293T cell lysates transfected with pCMV (vector) or pCMV-Flag-3CL; (middle) density analysis results of LRPAP1 expression levels, normalized using ImageJ relative to GAPDH; and (right) density analysis results of IFNAR1 expression levels, normalized using ImageJ relative to GAPDH.

[0056] Figure 33 The results of immunoblotting of RD cell lysates 24 hours after MOI infection with HSV-1 are described, along with density analysis of LRPAP1 expression levels, and normalized relative to GAPDH using ImageJ software.

[0057] Figure 34A Describes the viral RNA levels of HCoV-OC43 as analyzed by RT-qPCR. Figure 34B Description via TCID 50 The viral titer of HCoV-OC43 was determined experimentally. Figure 34C Describe the relative viral DNA levels of HSV-1 as analyzed by RT-qPCR. Figure 34D The results of Western blot analysis of HepAD38 cell lysates 48 hours after transfection with random siRNA or siLRPAP1 are described. Figure 34E Describes the ZIKV viral RNA levels as analyzed by RT-qPCR. Figures 34F to 34G This study demonstrates the inhibitory effect of anti-LRPAP1 antibody on HSV-1 infection.

[0058] Figure 35 Description (left) HepAD38 cell immunoblotting results used to validate HBV induction, with tetracycline (TET) used to inhibit integrated HBV genome transcription; (middle) Density analysis results of IFNAR1 expression levels, normalized using ImageJ relative to GAPDH; (right) Density analysis results of HBc expression levels, normalized using ImageJ relative to GAPDH.

[0059] Figure 36 The results of immunoblotting of ZIKV-infected RD cells (left) and density analysis of ZIKV envelope protein expression levels (right) are described, and normalized using ImageJ relative to GAPDH.

[0060] Figure 37The immunoblotting results of HepAD38 cell lysates (left) after 48 hours of transfection with pcDNA-LRPAP1 (OE) or 24 hours after treatment with rLRPAP1 (EX) are described, along with density analysis of IFNAR1 expression levels (right), normalized using ImageJ relative to GAPDH.

[0061] Figures 38A to 38B Immunoblotting results are presented to demonstrate the effect of α2M on EV71 or HSV-1 infection. Results are expressed as mean ± standard deviation (error bar) of three replicates. *P≤0.05, **P≤0.01, ***P≤0.001 (unpaired t-test).

[0062] Figure 39A Describe the immunoblotting results of lysates of HEK-293T cells after treatment with rLRPAP1 (200 nM), α2M (20 nM), or a combination of both proteins for 1 hour; Figure 39B The effect of lactoferrin (LF) on HSV-1 infection is shown (left).

[0063] Figure 40A The effect of lactoferrin (LF) on HSV-1 infection is demonstrated. Figure 40B Describe the immunoblotting results of lysates of HepAD38 cells 24 hours after treatment with LF (400 nM) or α2M (20 nM). Detailed Implementation

[0064] In the following description, LRPAP1 inhibitors (α2-macroglobulin, α2M) used as monotherapy, in combination with other types of antiviral drugs, or as drug carriers for delivering α2M for broad-spectrum antiviral therapy are described as preferred examples. It will be apparent to those skilled in the art that modifications, including additions and / or substitutions, can be made without departing from the scope and spirit of the invention. Certain specific details have been omitted from this invention to avoid confusion; however, the disclosure herein is sufficient to enable those skilled in the art to practice the invention without excessive experimentation.

[0065] Dysfunction or loss of low-density lipoprotein receptor-associated protein 1 (LRPAP1) is associated with a variety of neurodegenerative diseases, including degenerative dementia (such as Alzheimer's disease, atherosclerosis, etc.) and myopia. Increasing evidence suggests that these neurodegenerative diseases may be caused by a weakened innate immune response in the brain to chronic infections. Although positive-sense single-stranded RNA viruses (such as EV71, ZIKV, and SARS-CoV-2) complete their life cycle in the cytoplasm, and DNA viruses (such as HSV-1) replicate their genome in the cell nucleus, both viruses can infect nerve cells in the central nervous system and cause acute or chronic neurological diseases (such as paralysis, neurodegenerative diseases), and even death. After entering the cell and uncoating, the genomic RNA of positive-sense single-stranded RNA ((+)ssRNA) viruses is translated into polypeptide chains, which are then cleaved into functional proteins by viral proteases. Viral proteases play a crucial role in promoting viral replication through interactions, modifications, and cleavage with host or viral proteins.

[0066] EV71 2A protease (2A) pro EV712A protease (2A) is widely considered to be a major factor in reducing IFNAR1 levels in cells. pro Not only can it cleave host proteins to promote viral protein translation, but it can also interact with various signaling proteins to regulate the processing and translation of viral RNA. For example, EV71 infection and its encoded 2A... pro It can promote the secretion of LRPAP1 into the extracellular environment, thereby leading to a more than 5-fold decrease in the expression level of IFNAR1 protein on the cell surface. However, 2A pro The host protein itself cannot directly degrade IFNAR1, suggesting that the process may involve other host proteins, but it is currently unclear which specific host proteins are involved. Furthermore, 2A pro It can also inhibit the P-processor in the host cell and isolate its component proteins to promote the synthesis of viral RNA.

[0067] SARS-CoV-2 3CL protease (3CL) pro 3CL participates in the synthesis of viral proteins and is responsible for assembling viral proteins into mature functional proteins. pro It can also upregulate the expression and secretion of LRPAP1.

[0068] Interferon signaling pathways play a crucial role in broad-spectrum antiviral activity and as the first line of defense against viruses. The LRPAP1-dependent IFNAR1 degradation mechanism may serve as a universal pathway for both DNA and RNA viruses to circumvent host defenses. As a ligand, the N-terminus of secreted LRPAP1 binds to the extracellular domain of IFNAR1, inducing receptor ubiquitination and promoting its degradation, thereby enhancing viral infection in vitro, in isolated mouse brain tissue, and in newborn mice. Small peptides derived from the N-terminus of LRPAP1 can effectively bind to and induce IFNAR1 degradation, thereby enhancing the infectivity of DNA and RNA viruses, including herpesviruses (such as herpes simplex virus type 1 (HSV-1), hepatitis B virus (HBV)), EV71, and coronaviruses (such as HCoV-OC43). Currently, broad-spectrum antiviral drugs on the market are extremely limited. This invention provides an effective broad-spectrum antiviral method aimed at protecting IFNAR1 expression before or after viral infection.

[0069] This invention reveals a novel "outdoor" mechanism illustrating how viral stimulation-induced secretion of LRPAP1 acts as an inhibitory ligand, binding to IFNAR1 and inducing its degradation. Figure 1 This invention provides a method for preventing and / or treating viral infections. The method comprises administering an effective amount of an LRPAP1 inhibitor to a subject in need to target LRPAP1, thereby increasing the expression of interferon and its derivatives in the subject's body. This invention has found that silencing LRPAP1 reduces viral infection levels, including RNA viruses (such as pituitary RNA viruses, coronaviruses, and flaviviruses) and DNA viruses (such as herpesviruses and hepatitis viruses). These viruses infect nerve cells in the brain, leading to acute neuronal dysfunction or death, as well as chronic neurological disorders, which may be caused by a combination of acute and long-term immune responses.

[0070] In one embodiment, the LRPAP1 inhibitor is a pharmaceutical composition comprising α2-macroglobulin (α2M). α2M is a rich plasma protein produced by the liver, with a concentration of 240 to 290 mg / 100 mL in adult blood. It participates in clearing amyloid-β (Aβ) peptide from the brain and can reduce the level of cartilage-degrading factors. In fact, direct injection of α2M has been used in FDA-compliant clinical settings for the treatment of arthritis and other orthopedic-related conditions. 1

[0071] α2M blocks the binding of the N-terminus of secreted LRPAP1 to the extracellular domain of interferon α / β receptor 1 (IFNAR1). This blocking effect prevents LRPAP1 from binding to the extracellular domain of IFNAR1, thereby interrupting downstream signaling pathways associated with IFNAR1 activation. Consequently, the immune response mediated by interferon α and β is modulated, thereby affecting the activation of immune cells, the production of cytokines, and antiviral responses.

[0072] The pharmaceutical composition may also contain a pharmaceutically acceptable adjuvant. A pharmaceutically acceptable adjuvant may be an emulsifier, surfactant, stabilizer, preservative, or buffer.

[0073] In another embodiment, the LRPAP1 inhibitor is a siRNA with sequence number SEQ ID NO.: 1. Upon administration, the siRNA sequence enters the target cell and binds to the mRNA molecule encoding LRPAP1 through complementary base pairing.

[0074] In another embodiment, the LRPAP1 inhibitor is a lysosomal inhibitor. Lysosomes are intracellular organelles responsible for the degradation and recycling of cellular components, including proteins. Lysosomal inhibitors suggest a mechanism involving disruption of lysosomal degradation pathways.

[0075] This invention demonstrates that a specific region within domain 1 of LRPAP1 contains a key site for interaction with IFNAR1. More importantly, the synthetic peptide RAPD1P1 exhibits a potent ability to inhibit IFNAR1 and promote viral infection.

[0076] LRPAP1 assists in LRP maturation and acts as a potent antagonist of LRP on the cell membrane. Elevated LRPAP1 levels inhibit the extracellular domain of LRP, thereby hindering the binding and endocytosis of ligands (e.g., β-amyloid, LDL cholesterol rich in apolipoprotein B and apolipoprotein E). This invention confirms that in viral infection or viral proteases (e.g., 2A) pro and 3CL pro Following expression, LRP1 levels decreased, and this decrease was negatively correlated with an increase in LRPAP1 levels. This observation suggests that the interaction between the protease and LRPAP1 disrupts LRP1 maturation, triggering a compensatory response involving increased LRPAP1 production. LRP1 is known to activate the Notch signaling pathway, which is associated with various diseases, including cancer. The results showed that Notch levels decreased after EV71 or ZIKV infection.

[0077] In addition, multiple viral proteases were observed to have similar sequences, especially proteases within the same family.

[0078] The route of administration for LRPAP1 inhibitors can vary depending on the patient's condition and medical needs. Possible routes of administration include injection, topical application, intravenous injection, or other routes.

[0079] In summary, this invention elucidates the relationship between low-density lipoprotein receptor-associated protein 1 (LRPAP1) and 2A. pro The interaction between them. More importantly, this invention discloses a broad-spectrum antiviral method targeting LRPAP1. Results show that it has good antiviral efficacy within the biosafety range (<1.5 mg / 100 mL). Therefore, LRPAP1 inhibitors can be directly used clinically to enhance the therapeutic effect of interferon-dependent therapy or promote drug treatment for multiple viral infections.

[0080] Example

[0081] Example 1 - Materials and Methods

[0082] Cells and viruses

[0083] Rhabdomyosarcoma cells (RD), human embryonic kidney 293 cells containing SV40 large T antigen (HEK-293T), human adenocarcinoma alveolar basal epithelial cells (A549), African green monkey kidney epithelial cells (Vero), HBV-inducible HepG2 cells (HepAD38), and mouse breast cancer cells (4T1) were maintained in DuPont modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0084] EV71 (SHZH98 strain; GenBank accession number AF302996.1) was obtained from the Shenzhen Center for Disease Control and Prevention, China. HSV-1 (VR-1493) TM ), HCoV-OC43 (VR-1558) TM ) and ZIKV (VR-84) TM The virus was purchased from the U.S. Center for Type Culture Collection. To prepare the virus stock solution, the virus was proliferated in DMEM containing 2% FBS on a 90% confluent monolayer of cells.

[0085] plasmids and mutants

[0086] LRPAP1 was constructed using the pcDNA3.1 (Invitrogen) backbone for expression in mammalian cells, while pQTEV-LRPAP1 (Addgene) was used for recombinant protein expression. Ubiquitin and LRPAP1 mutants were constructed using the pCMV (Invitrogen) backbone. IFNAR1 and viral protein (2A, 3C, and 3D) expression vectors were constructed using the pcDNA4 / His B (Invitrogen) backbone. Each cDNA fragment was directly cloned into the Not I and Xba I sites of the vector. 2A... pro Cys 110 Mutation to Ala 110 (Get 2A) C110A The mutagenesis was performed using a one-step mutagenesis kit (Invitrogen). Primer sequences used for construction are available upon request. The SARS-CoV-2 gene was constructed using the pCDH-CMV backbone.

[0087] Recombinant protein purification

[0088] Expression constructs of protease 2A (pET28a-2A), 2A mutant (pET28a-2Am), or LRPAP1 (pQTEV-LRPAP1) were transformed into Lemo21 competent *E. coli* according to NEB protocols. The bacteria were cultured at 37°C until an OD600 of 0.4–0.6 was achieved. Subsequently, 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the bacteria were incubated overnight at 16°C to induce protein expression. Cell lysates were obtained using a French Press at 1000 kPa at 4°C. The lysates were collected by centrifugation at 20,000 rpm at 4°C. Protein purification was then performed according to the Ni-NTA purification protocol (QIAGEN). The purified protein was stored in a modified protein stabilization buffer (1 mM MTT, 0.5 mM EDTA, 50 mM NaCl, 50 mM Tris-HCl pH 8.0, 35% (v / v) glycerol) and kept at -20°C.

[0089] peptides

[0090] The extracellular domain of recombinant interferon receptor 1 (ECD-IFNAR1) was purchased from Sino Biological (catalog number: 13222-H08H). Peptides RAPD1P1 (YSREKNQPKPSPKRESGEE), RAPD1P2 (FRMEKLNQLWEKAQRLHLPPV), the out-of-order peptide (control ligand), and the cell-penetrating peptide RV5 (RGDFV) were synthesized by GenScript Biotech with a purity of 95%.

[0091] Animal research

[0092] C57BL / 6 mice were purchased from City University of Hong Kong and bred there. All animal experimental protocols were approved by the Ethics Committee of City University of Hong Kong and licensed by the Hong Kong Department of Health (No.: DH / SHS / 8 / 2 / 5 Pt.3, 18-69). This invention was carried out in accordance with the approved protocols.

[0093] Newborn C57BL / 6 mice are intraperitoneally injected weekly with EV71 (2×10⁻⁶). 8 Administer PFU or EV71+rLRPAP1 (200 nM) twice daily. Assess the neurological status and behavior of each mouse daily. Sacrifice mice on day 7 and collect intestinal and blood samples for virus detection.

[0094] In vitro and in vivo viral infections

[0095] After washing cells twice with PBS, they were infected with EV71 or a specified virus at the multiple of infection (MOI) shown in the diagram. One hour after adsorption, the inoculum was removed, and the cells were washed twice to remove unbound virus; then culture medium was added. In virus entry and uncoating experiments, the inoculum and cells were collected after specified time intervals.

[0096] In in vivo experiments, newborn mice were intraperitoneally injected weekly with rLRPAP1 (200 nM) and EV71 (2 × 10⁻⁶ mcg). 8 PFU) or EV71 with rLRPAP1 twice.

[0097] Immunoblotting

[0098] Total cellular protein was prepared using radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1× Roche protease inhibitor mixture, 1× Roche Phostop) under intermittent vortex mixing conditions. The protein samples were then analyzed by Western blotting. In the immunoblotting analysis, the proteins were detected using specific antibodies against VP1 (Abnova, PAB7631-D01P), ZIKV-Env (GeneTex, GTX133314), HSV-Env (Invitrogen, PA5-38569), HBx (Santa Cruz Biotechnology, sc-57760), HBs (EXBIO, 11-329-C100), HBc (Santa Cruz Biotechnology, sc-23947), LRP1 (Abcam, ab92544), LRPAP1 (Abcam, ab14404), IFNAR1 (Abcam, ab45172), eIF4G (Santa Cruz Biotechnology, sc-11373), and ubiquitin (Santa Cruz Biotechnology, sc-8017). The target protein was detected by the corresponding secondary antibody (Santa Cruz Biotechnology) and finally subjected to colorimetric imaging using a chemiluminescence detection system (Amersham Biosciences).

[0099] mRNA extraction and qRT-PCR

[0100] Cellular mRNA was extracted according to the operating procedures of RNAiso Plus (TaKaRa, catalog number: 9108). Total RNA was reverse transcribed into cDNA using a one-step reverse transcription system (Takara, catalog number: RR024A). Quantitative reverse transcription PCR (qRT-PCR) was performed using an ABI 7500 real-time PCR system and SYBR Green Master Mix (Applied Biosystems). PCR was performed under the following thermal cycling conditions: 45 cycles, each consisting of 30 seconds at 95°C and 5 seconds at 60°C. Fluorescence signals were acquired by the instrument during the extension phase of each PCR cycle. The threshold cycle number (CT value) for each protein was normalized to the CT value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). qRT-PCR was performed using the primer pairs shown in Table 2.

[0101] Table 2 - Primers for Specified Genes

[0102] Immunofluorescence imaging

[0103] The following steps were performed to detect the cellular expression of IFNAR1, viral protein VP1, and LRPAP1 in HEK-293T cells with or without EV71 stimulation, under recombinant LRPAP1 treatment, and under LRPAP1 or 2A overexpression conditions.

[0104] In summary, HEK-293T cells were washed twice with PBS and fixed with 4% paraformaldehyde for 20 minutes. Subsequently, the cells were incubated for 2 hours in blocking buffer (PBS containing 10% (v / v) FBS, 1% (v / v) BSA, and 0.1% (v / v) Triton). The cells were then incubated with specific antibodies. The viral structural proteins VP1, LRPAP1, and IFNAR1 were detected using specific antibodies against VP1 (Abnova, PAB7631-D01P), LRPAP1 (Atlas, HPA008001), and IFNAR1 (Novus, NBP1-83119). After washing cells three times with PBST (PBS + 0.1% Tween-20), cells were labeled by incubating for 1 hour at 4°C with a secondary antibody conjugated to Alexa Fluor 488 (green) or Alexa Fluor 594 (red) (dilution 1:1000) (Life Technologies). After washing cells three times with PBST (PBS + 0.1% Tween-20), cell nuclei were stained with DAPI (0.5 μg / mL) for 5 minutes at room temperature. Finally, cells were fixed onto slides using Prolong Gold anti-quenching mounting medium. Imaging was performed using a Carl Zeiss LSM 880 confocal microscope with a 63× oil immersion lens, and analysis was conducted using ZEN software.

[0105] For live-cell imaging, HEK-293T cells were transfected with pcDH-eGFP-IFNAR1 for 48 hours. Subsequently, they were treated with the cell membrane dye CellMask (C10046, Invitrogen). TM ) and nuclear dye Hoechst 33342 (62249, ThermoScientific TM Cells were pre-stained. Subsequently, cells were pretreated on ice with PBS or 200 nM rLRPAP1 for 30 minutes. Live cells were continuously recorded for 1 hour in a Thermo Fisher Cell Insight (CX7) system connected to a CO2 device and maintained at 37°C.

[0106] Immunoprecipitation

[0107] The interaction between LRPAP1, LRP1, and IFNAR1 was detected using the immunoprecipitation method described below. HEK293T cells were cultured in 100 mm tissue culture dishes with approximately 90% confluence. After washing the cells with 10 mL PBS, they were collected in 0.5 mL of ice-cold lysis buffer (0.15 mM NaCl, 0.05 mM Tris-HCl (pH 7.4), 1% SDS, 1% NP-40, a mixture of protease inhibitors (Roche), and PhoSTOP (Roche)). The total extract was pre-cleaned by incubating 10 μg of normal rabbit IgG (Cell Signaling, 2729S) and 20 μL of protein A / G complex agarose (Santa Cruz Biotechnology) on a rotor at 4 °C for 1 hour. After gently removing the protein A / G agarose beads by centrifugation at 500 rcf for 2 minutes, 0.2 mg of the total extract and the specified antibody were incubated on a rotor at 4 °C overnight for immunoprecipitation. Approximately 20 μL of Protein A / G complex agarose (Santa Cruz Biotechnology) was added and incubated at 4 °C for 1 hour. Subsequently, the magnetic beads were washed three times with washing buffer (lysis buffer containing 0.1% NP-40). The precipitate was resuspended in 40 μL PBS and 10 μL 5×SDS-PAGE loading buffer, and the sample was boiled for 10 minutes. The supernatant was collected, and 20 μg of the lysis buffer was loaded onto a 10% polyacrylamide gel for electrophoresis. Immunoblot analysis was performed using antibodies against IFNAR1 (Abcam, ab45172), LRP1 (Abeam, ab92544), and LRPAP1 (Abcam, ab14404).

[0108] Protein docking

[0109] The experimentally determined three-dimensional structures of LRPAP1 (2p03) and IFNAR1 (3s98) were obtained from the Protein Database (PDB). Protein-protein docking predictions were performed using H-dock (http: / / hdock.phys.hust.edu.cn / ). Docking models were selected based on confidence scores, with scores above 0.7 indicating extremely high binding potential between the two proteins.

[0110] Flow cytometry assay

[0111] Mouse breast cancer cells (4T1) were stained on ice with APC-conjugated IFNAR1 antibody (Biolegend, #127314, 1:100) for 30 minutes. Cells were then treated with or without rLRPAP1 (200 nM) for specified times. Fluorescence-activated cells were sorted and analyzed using a Beckman Coulter CytoFLEX S flow cytometer (Becton Dickinson). Results were analyzed using FlowJo (version 10.5.3).

[0112] Separating membrane proteins from cytoplasmic proteins

[0113] After washing the tissue twice with PBS, a homogenate was prepared using a 2 mL homogenizer. Membrane proteins were then separated from cytoplasmic proteins according to the operating procedure of the membrane protein extraction kit (Thermo, 89842).

[0114] Micro-thermophoretic (MST) analysis

[0115] Multihistidine (His)-tagged peptides, recombinant 2A, recombinant 2Am, and ECD-IFNAR1 were incubated with RED-tris-NTA second-generation dye (catalog number MO-L018, Nanotemper-Technologies) for 30 minutes. Subsequently, these fluorescently labeled peptides were mixed with serially diluted ligands, including out-of-order peptides, commercially available penetrant peptides (RV5), LRPAP1 peptides (RAPD1P1, RAPD1P2), or His-free rLRPAP1. The mixtures were then loaded into Monolithic containers. TM The assay was performed using NT.115 series capillary tubes (part number MO-K022). The capillary tubes were then placed in a Monolith NT system for MST analysis. According to Monolith guidelines, a signal-to-noise ratio (S / N) greater than 5.0 indicates the presence of effective binding.

[0116] Statistical analysis

[0117] Results are expressed as mean ± standard deviation (SD). Two-tailed Student's t-tests were used for comparisons between two groups, and a p-value <0.05 was considered statistically significant.

[0118] Example 2 - Enhancement of viral infection by EV71-induced increase in LRPAP1 expression

[0119] To identify with EV712A pro Interacting proteins, this invention uses recombinant 2A pro (r2A) and protease-inactivated 2A C110A The mutant (r2Am) was used as a decoy for mass spectrometry (MS) and micro-thermophoretic (MST) analysis.

[0120] See Figure 2A Reorganization of 2A pro The protein obtained from the (r2A) pull-down assay was separated by one-dimensional PAGE electrophoresis and visualized by silver staining. The bands shown were excised and analyzed by MS. The results indicated that LRPAP1 and 2A... pro Interaction occurs ( Figure 2A (and Table 1). LRPAP1 can transport low-density lipoprotein receptor-associated protein (LRP) from the endoplasmic reticulum (ER) to the Golgi complex and act as an effective antagonist of LRP on the cell surface.

[0121] Table 1 - Proteins identified by mass spectrometry

[0122] Figure 2B The display shows 2A pro Induced increases in LRPAP1 expression lead to decreased LRP1 protein levels. Interestingly, although 2A pro It can effectively cut eIF4G, but it cannot directly cut LRPAP1 ( Figure 2C ). 2A pro (instead of 2Am) and EV71 ( Figure 2D-2E LRPAP1 expression at both mRNA and protein levels was increased in both RD cells and HEK293T cells, while other viral proteins (such as 3C) showed increased expression. pro 3D(RdRp)) does not have this effect. Figure 2F ).

[0123] Furthermore, ectopic expression of LRPAP1 significantly promoted EV71 infection, while knockdown of LRPAP1 significantly reduced EV71 replication and spread by more than 80%, and consistent results were obtained using two other siRNAs. Figures 3A-3B ).

[0124] Example 3 - Enhancement of viral infection by SARS-CoV-2-induced increase in LRPAP1 expression

[0125] To investigate whether proteases encoded by different viruses have similar functions, this invention uses EV712A... pro The sequence was compared with other viral protease sequences. Surprisingly, high sequence conservation was found in proteases from the Picornaviridae family, including rhinovirus (RV-C) and enterovirus (Coxsackievirus A16). Furthermore, it was also found in EV71 2A. pro With coronavirus 3CL pro High similarity was found between (MERS-CoV and SARS-CoV-2) and flavivirus proteases (Zika virus and dengue virus). Figure 4These findings suggest a potential synergistic effect of multiple viral proteases on LRPAP1.

[0126] Similar to EV71, SARS-CoV-2 can also cause severe neurological disorders and even death. Previous studies have shown that coronavirus proteases are similar to enterovirus 2A. pro The similarity between them can be used to suppress innate immunity. 1-4 In addition to comparable amino acid sequences, enterovirus 2A was also detected. pro With SARS-CoV-2 3CL pro There are similar three-dimensional structures between them. Figure 5 Therefore, this invention utilizes coronavirus 3CL. pro As a representative study, we further investigated the effects of various proteases on LRPAP1 and viral infection. Interestingly, SARS-CoV-2 3CL... pro It can also increase LRPAP1 expression, while PL pro Then it will not have this effect. Figure 6A In addition, 3CL pro These are evolutionarily conserved proteins among different coronaviruses. The 3CL protein is located between SARS-CoV-2 and HCoV-OC43. pro High similarity was observed in the protein amino acid sequences. Furthermore, elevated LRPAP1 expression was detected after HCoV-OC43 infection, suggesting that coronaviruses may play a similar role in promoting LRPAP1 expression. Figure 6B ).

[0127] Example 4 - Secretory LRPAP1 assists in coronavirus and EV71 infection

[0128] When the intracellular LRP transport system produces excess LRPAP1, it is secreted into the extracellular environment. This invention further confirms that, in the expression of SARS-CoV-2 3CL... pro In cells, extracellular LRPAP1 levels are elevated ( Figure 7 Subsequently, the fold change of extracellular RPAP1 in the culture medium was measured by ELISA.

[0129] See Figure 8A RD cells were infected with HCoV-OC43 at MOI 2 for 72 hours. The supernatant was collected and treated with either 0.5 μg / mL anti-LRPAP1 antibody (anti-LRPAP1) or IgG for 1 hour. This supernatant was then used to infect new RD cells for 12 hours. Extracellular LRPAP1 was also collected after HCoV-OC43 infection. Treatment with LRPAP1 antibody reduced intracellular and extracellular viral RNA levels by approximately 40% and 70%, respectively. Figure 8B ).

[0130] See Figure 8C RD cells were infected with HCoV-OC43 at MOI2 for 72 hours. The supernatant was collected and treated with either 0.5 μg / mL anti-LRPAP1 antibody (anti-LRPAP1) or IgG for 1 hour. This supernatant was then used to infect new RD cells for 12 hours. LRPAP1 antibody treatment also significantly protected cells from viral infection-induced cytopathic effects (CPE), indicating that HCoV-OC43-induced extracellular LRPAP1 promotes viral infection.

[0131] also, Figure 9 The results showed that RD cells were infected with EV71 at MOI10 for 3 hours. Subsequently, the cells were treated with or without anti-LRPAP1 antibody (anti-LRPAP1) for 6 hours. Adding LRPAP1 antibody to the culture medium 3 hours post-infection significantly reduced the EV71-induced cytopathic effect. ELISA detection showed that EV71-infected cells and 2A... pro Elevated extracellular LRPAP1 levels were detected in all expression cells, while in 2A... C110A No expression was detected in the cells ( Figures 10A-10B More importantly, supplementing the culture medium with rLRPAP1 significantly promoted EV71 infection and EV71-induced cytopathic effects. Figure 10C-10D ).

[0132] See Figure 11A HEK-293T cells were pretreated with rLRPAP1 on ice for 1 hour. Subsequently, cells were seeded with EV7 at MOI 50 for 15 minutes or 1 hour. Intracellular viral particles were collected and then subjected to viral titration. Figure 11B ).exist Figure 11C In this study, the lysate was immunoblotted using anti-VP1 and anti-LRPAP1 antibodies. Short-term extracellular exposure to rLRPAP1 enhanced the entry and uncoating efficiency of EV71 in HEK-293T cells within 1 hour.

[0133] Subsequently, the role of rLRPAP1 in the EV71-infected C57BL / 6 mouse model was further investigated. See also Figure 12 C57BL / 6 mice were intraperitoneally injected twice weekly with either EV71 (2 × 10⁸ PFU) or EV71 + rLRPAP1 (200 nM). Mice were sacrificed on day 7, and their intestines and blood were collected for virus detection. The number of mice successfully infected was determined by RT-qPCR analysis of serum samples. A Ct value less than 38 was considered a successful infection; otherwise, the mice were considered uninfected. Surprisingly, rLRPAP1 significantly enhanced EV71 infection and exacerbated disease in reborn C57BL / 6 mice. Figure 12-13The results showed that the expression of both LRPAP1 and VP1 proteins was significantly increased after the addition of rLRPAP1. Furthermore, C57BL / 6 mice co-injected with EV71 and rLRPAP1 exhibited severe physiological dysfunction after injection. Figures 14A-14B The results in summary indicate that secretory LRPAP1 plays a crucial role in promoting viral infection both in vitro and in vivo.

[0134] Example 5 - Extracellular LRPAP1-dominantly drives the downregulation of IFNAR1

[0135] Since LRPAP1 antibodies significantly inhibited coronavirus activity, it is speculated that secreted LRPAP1 may act as an inhibitory ligand for IFNAR1. See also Figure 15 HEK-239T cells were transfected with non-specific control siRNA or siLRPAP1 for 24 hours, followed by transfection with empty vector or CMV-flag-3CL for an additional 24 hours. Results showed that in wild-type HEK-239T cells, 3CL showed significantly higher activity compared to lanes 1 and 3. pro LRPAP1 expression was increased and IFNAR1 levels were decreased. However, IFNAR1 expression was increased in LRPAP1 knocked-down cells (lanes 1 and 2). Interestingly, IFNAR1 expression was increased in cells with 3CL knockdown. pro The transfection-induced decrease in IFNAR1 levels was restored after LRPAP1 silencing, which is consistent with this hypothesis.

[0136] Furthermore, for only 2A pro Ectopic expression and LRPAP1 and 2A pro Simultaneously, the levels of ectopically expressed IFNAR1 and LRPAP1 were compared. RD cells were transfected with pcDNA3.1 (vector) or pcDNA-LRPAP1 for 24 hours, and then infected with EV71 at an MOI of 10 for 9 and 12 hours. Immunoblot analysis was performed using the corresponding antibodies against IFNAR1, LRPAP1, and the viral structural protein VP1. IFNAR1 levels were quantified using ImageJ density analysis and normalized using GAPDH. Figures 16A-16E The display shows that the EV71 and 2A pro LRPAP1 expression was promoted and IFNAR1 levels were reduced, with LRPAP1 alone reducing IFNAR1 to near-detectable minimum levels in HEK-293T cells. Furthermore, LRPAP1 expression was correlated with 2A... pro The synergistic effect of these mechanisms makes IFNAR1 levels completely undetectable. Figure 16C-16D ).

[0137] Conversely, HEK-293T cells were first transfected with non-specific control siRNA (NC) or siLRPAP1 for 24 hours. Then, cells were transfected with an empty vector or 2A expression plasmid for another 24 hours, or infected with EV71 for a specified time. IFNAR1 levels were quantified using ImageJ density analysis and normalized using GAPDH. Results showed that LRPAP1 knockdown alleviated 2A... pro Induced IFNAR1 downregulation Figures 17A-17B ).

[0138] Excessive LRPAP1 expression inhibits the type I interferon signaling pathway and its downstream interferon-stimulated gene (ISG). Figure 18A LRPAP1 knockdown enhances ISG expression, thereby promoting EV71 infection. Figure 18B The above results indicate that LRPAP1 plays a role in the 3CL of SARS-CoV-2. pro and Enterovirus 71 2A pro It plays a key role in the downregulation of IFNAR1 and interferon signaling pathways.

[0139] The effect of secreted LRPAP1 on IFNAR1 reduction was also reassessed using LRPAP1 antibody treatment. EV71 cells were infected with an MOI of 10 for 3 hours. Cells were then treated with or without anti-LRPAP1 antibody (anti-LRPAP1) for 6 hours. Lysates were immunoblotted using anti-IFNAR1, anti-EV71 VP4, and anti-LRPAP1. Figure 9 Consistent with CPE detection results, IFNAR1 levels successfully returned to normal after LRPAP1 antibody treatment following EV71 infection. Surprisingly, the LRPAP1 antibody also reduced intracellular LRPAP1 and viral protein VP4 levels. Figure 19A To demonstrate the major role of extracellular LRPAP1 in reducing IFNAR1, HEK-293T cells were treated with different concentrations of rLRPAP1 for a maximum of 15 minutes or 1 hour. See also... Figure 19B IFNAR1 decreased rapidly in a dose-dependent manner within 15 minutes in response to rLRPAP1. Within just one hour of treatment with rLRPAP1, IFNAR1 levels decreased to near-detectable minimum at a concentration of 50 nM, and to undetectable levels at a concentration of 200 nM. This further indicates that extracellular LRPAP1 downregulates IFNAR1.

[0140] Example 6 - LRPAP1 triggers [the following] in vitro and in vitro brain tissue by binding to the extracellular domain of IFNAR1. Degradation of IFNAR1

[0141] To investigate how extracellular LRPAP1 interferes with IFNAR1 expression in the early stages of viral infection, protein levels in membrane and cytoplasmic components were compared between untreated cells and cells treated with 200 nM rLRPAP1 for 1 to 10 minutes.

[0142] Figure 20A The results showed that IFNAR1 decreased continuously over time, while rLRPAP1 gradually accumulated in the membrane components. Furthermore, IFNAR1 and LRPAP1 were highly co-localized on the cell membrane and significantly decreased, a phenomenon observed after EV71 infection where LRPAP1 increased sharply. Figure 20B ) or processed by rLRPAP1 ( Figure 20C It was implemented later.

[0143] In the brain, innate immunity in neurons plays a major role in limiting or clearing viral infections because nerve cells are the host cells for viruses, and their adaptive immunity is relatively low. Infections with various viruses (SARS-CoV-2, ZIKV, DENV, EV71, etc.) can cause severe neurological disorders and even death, such as aseptic meningitis, encephalitis, polio-like paralysis, and mental disorders. To examine whether LRPAP1 promotes viral infection in brain nerve cells, the effect of extracellular rLRPAP1 on membrane-bound IFNAR1 was verified using mouse brain tissue under in vitro conditions.

[0144] See Figure 21A Freshly prepared brains from 8-week-old C57BL / 6 mice were divided into two hemispheres and immediately incubated in oxygenated cuvettes containing either freshly prepared artificial cerebrospinal fluid (ACSF) (127 mM NaCl, 1 mM KCl, 1.2 mM KH2PO4, 26 mM NaHCO3, 10 mM D-glucose) or ACSF containing 200 nM rLRPAP1 at 0°C for 1 hour. Subsequently, immunoblotting analysis was performed on the cytoplasmic and membrane components of the brain tissue. Compared with the control hemisphere from the same mouse, the IFNAR1 level in the rLRPAP1-treated hemisphere was significantly reduced (…). Figure 21B ).

[0145] To more clearly depict the interaction between rLRPAP1 and IFNAR1 on the cell surface, the extracellular domain of APC-labeled IFNAR1 was detected by flow cytometry in live 4T1 cells treated with and untreated with rLRPAP1, respectively. Extracellular rLRPAP1 significantly reduced the level of IFNAR1 on the cell surface. Figure 22AFurthermore, HEK-293T cells were transfected with pcDNA3.1 (vector) or pcDNA-LRPAP1, or treated with non-specific control siRNA (NC) or siRNA-LRPAP1 (siLRPAP1) for 48 hours. It was also noted that LRPAP1 had no effect on IFNAR1 mRNA levels with or without interferon treatment. Figure 22B In summary, extracellular rLRPAP1 directly downregulates IFNAR1 on the cell membrane without affecting IFNAR1 transcription or mRNA stability.

[0146] This invention further utilizes a co-immunoprecipitation experiment ( Figure 23A The interaction between LRPAP1 and IFNAR1 was confirmed. Furthermore, mouse brain tissue was homogenized using a 2 mL tissue homogenizer and immunoprecipitated with antibodies against LRPAP1 and IFNAR1, respectively. Their binding was also observed in the mouse brain. Figure 23B Furthermore, pull-down experiments showed that rLRPAP1 can directly bind to the extracellular domain of IFNAR1. Figure 23C ), using molecular docking to predict its potential binding regions, such as Figure 23D As shown. The peptides RAPD1P1 and RAPD1P2 derived from the N-terminus of LRPAP1 were tested, with RAPD1P1 showing binding potential while RAPD1P2 did not.

[0147] Immunoblot analysis was performed on lysates of RD cells treated with either a non-specific control peptide (control) or a specific LRPAP1 peptide (1 μM) for 1 hour. RD cells were treated with 1000 U / mL IFN-α2b and a specified peptide (control or RAPD1P1) for 1 hour. Downstream responses of the type I interferon signaling pathway were assessed by the relative mRNA levels of specific ISGs. Figures 24A-24B The results showed that RAPD1P1 significantly reduced the levels of IFNAR1 and its downstream antiviral ISG. See also... Figure 25 RD cells were pretreated with a specified peptide for 1 hour, and then seeded for 9 hours with or without EV71 (MOI = 10). EV71 CPE was characterized by cell rounding and detachment. Scale bar: 100 μm (left image). Subsequently, the viral titer of the supernatant from EV71-infected cells was determined (right image). Similar to rLRPAP1, RAPD1P1 also significantly promoted EV71 infection. Importantly, microscale thermophoresis (MST) showed that RAPD1P1, but not RAPD1P2, could bind to the extracellular domain of IFNAR1 (Kd = 11.3 nM). Figure 26 ).

[0148] The degradation of IFNAR1 begins with its phosphorylation, followed by endocytosis. Subsequently, phosphorylated IFNAR1 (p-IFNAR1) undergoes ubiquitination and is translocated to lysosomes for degradation. Cells were double-stained with the corresponding antibodies for EEA1 (red) and IFNAR1 (green). Cell nuclei were stained blue with DAPI. Scale bar: 5 μm. Figure 27A The study showed that the binding of rLRPAP1 to IFNAR1 induces IFNAR1 endocytosis, as revealed by detecting increased levels of the early endosome marker (EEA1). Furthermore, phosphorylated IFNAR1 was found to increase in a dose-dependent manner with respect to extracellular rLRPAP1. Figure 27B IFNAR1 ubiquitination was also upregulated by LRPAP1, indicating that LRPAP1 induces IFNAR1 degradation. Figure 27C ).

[0149] Furthermore, LRPAP1-induced IFNAR1 decrease can be mitigated by lysosomal inhibitors (including methylamine (MA)). Figure 28A ) and bafimycin ( Figure 28B It is blocked by the proteasome inhibitor MG132 or leucine, but is not affected by the proteasome inhibitor MG132 or leucine.

[0150] Because LRPAP1 significantly increased the ubiquitination of IFNAR1, and IFNAR1 was protected from degradation after MA treatment ( Figure 29 The above results collectively indicate that LRPAP1, as a ligand of IFNAR1, induces lysosome-dependent IFNAR1 degradation.

[0151] Example 7 - The N-terminus of LRPAP1 promotes infection by various viruses (DNA and RNA viruses).

[0152] RD cells were pretreated for 1 hour with a non-specific control peptide (control), RAPD1P1, RAPD1P2, or rLRPAP1. Subsequently, cells were infected with HCoV-OC43 at an MOI of 1 for 72 hours. RNA levels in HCoV-OC43 were detected by RT-qPCR. TCID43 levels were then analyzed. 50 The viral titer of HCoV-OC43 was experimentally determined. Both rLRPAP1 and RAPD1P1 treatments confirmed an increase in HCoV-OC43 RNA replication and viral proliferation. Treatment with rLRPAP1 or RAPD1P1 significantly increased intracellular RNA levels and viral titers, while RAPD1P2 had no such effect. Figures 30A-30B ).

[0153] Cells were pretreated with 1 μM of a specified peptide or 200 nM rLRPAP1 for 1 hour, then inoculated with the specified virus and cultured for the corresponding time (24 hours for HSV-1; 72 hours for HCoV-OC43). CPE in HSV-1 was characterized by cell rounding and detachment, while CPE in HCoV-OC43 was determined by intracytoplasmic vacuolation. The cytopathic effect induced by HCoV-OC43 infection was more pronounced after treatment with LRPAP1 and RAPD1P1. Figure 30G ), and then tested for coronavirus 3CL. pro The effects of induced secretory LRPAP1 on other viral infections ( Figure 31 293T cells were transfected with pCMV-SARS-CoV-2-3CL for 48 hours. The supernatant was collected and incubated with 0.5 μg / mL LRPAP1 antibody or IgG for 1 hour. The supernatant was then added to RD cells and incubated for 1 hour, followed by infection with MOI20 for 7 hours to detect EV71. Treatment with LRPAP1 antibody reduced both intracellular and extracellular EV71 RNA levels by 30%, and CPE was not significantly observed. Figure 30C-30E ).

[0154] 3CL was subsequently confirmed. pro Correlation with IFNAR1. Due to 3CL pro Induced increase in LRPAP1, decreased expression of IFNAR1 ( Figure 32 ).

[0155] This raises the question: does the upregulation of LRPAP1 expression and secretion represent a common strategy for viruses to evade the host cell's innate immunity? Besides RNA viruses (non-enveloped virus EV71 and enveloped coronavirus HCoV-OC43), the results showed that the DNA virus herpesvirus HSV-1 also promotes LRPAP1 expression. Figure 33 ).

[0156] Treatment with LRPAP1 and RAPD1P1 resulted in a more pronounced cytopathic effect induced by HSV-1 infection. Figure 30G RD cells were pretreated for 1 hour with a non-specific control peptide (control), RAPD1P1, RAPD1P2, or rLRPAP1. Subsequently, HSV-1 was infected at an MOI of 10 for 24 hours, and HSV-1 DNA levels were detected by RT-qPCR. More significantly, this invention found that LRPAP1 and RAPD1P1 promote HSV-1 DNA replication. Figure 30F ).

[0157] Subsequently, it was further explained that the SARS-CoV-2 3CL proThe effect of induced extracellular LRPAP1 on HSV-1 infection ( Figure 31 293T cells were transfected with pCMV-SARS-CoV-2-3CL for 48 hours. The supernatant was collected and incubated with 0.5 μg / mL LRPAP1 antibody or IgG for 1 hour. The supernatant was then added to RD cells and incubated for 1 hour, followed by infection with HSV-1 at MOI 20 and cultured for 7 hours. Intracellular HSV-1 RNA levels were detected by RT-qPCR and analyzed using TCID50. 50 The viral titer of HCoV-OC43 was determined. CPE of HSV-1 was characterized by cell rounding and shedding. Scale bar: 20 μm. After adding LRPAP1 antibody, intracellular HSV-1 DNA levels and TCID were observed. 50 Both decreased, and CPE was not significant in HSV-1 infection. Figure 30H-30J These results indicate that enhancing viral infection by promoting extracellular LRPAP1 is a conserved strategy applicable to both non-enveloped and enveloped viruses with RNA or DNA genomes.

[0158] Example 8 - Inhibition of multiple viral infections by inhibiting LRPAP1

[0159] Subsequently, this invention explored whether a broad-spectrum antiviral effect could be achieved by inhibiting or removing LRPAP1. See also Figures 34A-34B RD cells were transfected with non-specific control siRNA or siLRPAP1 for 24 hours. Subsequently, cells were infected with HCoV-OC43 at MOI 1 for 72 hours, or with HSV-1 at a specified MOI for 24 hours. Viral RNA levels in HCoV-OC43 were analyzed by RT-qPCR. TCID43 was used. 50 The viral titer of HCoV-OC43 was determined. The relative viral DNA level of HSV-1 was analyzed by RT-qPCR. With LRPAP1 knockdown, a significant decrease in HCoV-OC43 RNA level and viral titer was observed, while CPE was not significant. Similar results were obtained in HSV-1 infection. Figure 34C Furthermore, using the HepAD38 cell line, which stably expresses HBV, upregulation of IFNAR1 expression and a significant decrease in HBV (a DNA retrovirus) levels were detected in LRPAP1 knockdown cells. Figure 34D and Figure 35RD cells were transfected with non-specific control siRNA or siLRPAP1-1 / -2 (si-1, si-2) for 24 hours. Cells were then infected with MOI1 and cultured for 24 hours. The viral RNA level of ZIKV was analyzed by RT-qPCR. Interestingly, with LRPAP1 knockdown, the RNA level of ZIKV (an RNA flavivirus) decreased by more than 60%. Figure 34E ).

[0160] RD cells were transfected with nonspecific control siRNA or siLRPAP1 for 24 hours, and then inoculated with ZIKV at a designated MOI for 48 hours. Cell lysates were collected and immunoblotted using anti-IFNAR1, anti-LRPAP1, and ZIKA-Env. Following ZIKV infection, increased IFNAR1 expression and decreased viral load were observed in the knockdown cells. Figure 36 ).

[0161] Similarly, LRPAP1 overexpression resulted in decreased IFNAR1 levels and increased HBV load. Figure 37 It is hypothesized that a broad-spectrum antiviral strategy can be achieved by targeting LRPAP1. RD cells were infected with HSV-1 at MOI 20 for 13 hours. Subsequently, the cells were treated for 6 hours with or without 0.5 μg / mL LRPAP1 antibody (α-LRPAP1). Morphological characteristics of HSV-1-infected RD cells with or without anti-LRPAP1 treatment were observed, with CPE of HSV-1 manifested as cell rounding and detachment. Scale bar is 100 μm. Immunoblotting analysis was performed on RD cell lysates with or without LRPAP1 antibody treatment under HSV-1 infection conditions. This invention found that LRPAP1 antibody has a significant inhibitory effect on HSV-1 infection, as detected by less pronounced CPE and a decrease in viral protein levels. Figure 34F-34G ).

[0162] Furthermore, LRPAP1 is known to compete for binding with other ligands in the extracellular environment, including α2-macroglobulin (α2M) and lactoferrin (LF). 5-8 α2M and LF are both secretory proteins that are widely found in the blood, cerebrospinal fluid, and other bodily fluids of mammals.

[0163] RD cells were infected with EV71 or HSV-1 at MOI 10 for 13 hours, and then treated for the corresponding time with or without 20 nM α2M (6 hours for EV71; 21 hours for HSV-1). The density levels of VP1 and HSV envelope proteins were quantified using ImageJ and normalized with GAPDH. This invention validates that α2M, but not LF, significantly upregulates IFNAR1 under viral infection or rLRPAP1 treatment conditions. Figures 38A-38B See also Figure 39A and Figure 39B RD cells were infected with HSV-1 at MOI 10 and cultured for 6 hours. The cells were then treated with 400 nM LF for 18 hours. (Right image) shows the immunoblot of lysates from HepAD38 cells treated with LF (400 nM) or α2M (20 nM) for 24 hours. More importantly, α2M significantly inhibited the proliferation of both DNA and RNA viruses. Therefore, it can be concluded that inhibiting LRPAP1 can reduce infection by various viruses, not only RNA viruses (coronaviruses, enteroviruses, flaviviruses), but also DNA viruses (herpesviruses and hepatic DNA viruses). Enterovirus 2A pro Both LRPAP1 and SARS-CoV-23CLpro promote the production and secretion of the host protein LRPAP1, assisting viral infection by targeting the innate immune response. Overexpression of LRPAP1 can inhibit the extracellular domain of the LRP receptor, thereby hindering the binding and endocytosis of ligands such as amyloid-β protein and apolipoprotein E-rich low-density lipoprotein cholesterol. 9 In viral infection or 2A pro In cases of overexpression, LRPAP1 levels did indeed increase, accompanied by a decrease in LRP1 expression. This result indicates that 2A pro Interacting with LRPAP1 interferes with the maturation of LRP1, thereby triggering a compensation mechanism for the continuous generation of LRPAP1.

[0164] Furthermore, previous studies have reported that the LRP1-mediated Notch signaling pathway is highly conserved, and its dysfunction is associated with various diseases, including cancer. RD cells were infected with HSV at an MOI of 10 for 16 hours, followed by culturing with 400 nMLF for another 18 hours. In this invention, EV71 or ZIKV infection was also found to lead to a decrease in Notch-related protein levels. Figures 40A-40B This study not only confirmed the association between LRP1 and the Notch signaling pathway, but also provided new insights into the link between viral infection and the Notch signaling pathway.

[0165] The above description of the present invention is for illustrative and explanatory purposes only and is not intended to exhaustively describe the invention or limit it to the specific forms disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0166] definition

[0167] In this invention, the term "low-density lipoprotein receptor-associated protein 1 (LRPAP1)" is also referred to as α2-macroglobulin receptor-associated protein, 39kDa receptor-associated protein, low-density lipoprotein receptor-associated protein 1, or low-density lipoprotein receptor-associated protein 1 (α2-macroglobulin receptor-associated protein 1).

[0168] In this invention, the term "α2-macroglobulin (α2M)" is also referred to as α2-macroglobulin, protein containing C3 and PZP-like α2-macroglobulin domains 5.

[0169] Throughout this specification, unless the context otherwise requires, the word "comprise" or variations thereof, such as "comprises" or "comprising," should be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes. It should also be noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," and "comprising" may have the meaning conferred upon them under patent law; for example, they may permit elements not expressly stated, but exclude elements found in the prior art or affecting the essential or novel features of the invention.

[0170] Furthermore, throughout the specification and claims, unless the context otherwise requires, the word “include” or variations such as “includes” or “including” should be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes.

[0171] Unless otherwise defined, the terms “substantially,” “substantial,” “approximately,” and “about” as used herein are used to describe and explain small variations. When used in connection with an event or situation, these terms include both cases where the event or situation occurred precisely and cases where it approximately occurred. For example, when used in connection with numerical values, these terms may cover a range of variation of the value less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0172] References to "an embodiment," "an example embodiment," "example embodiment," etc., in this specification indicate that the described embodiment may include specific features, structures, or characteristics, but not every embodiment necessarily includes specific features, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same embodiment. Moreover, when a specific feature, structure, or characteristic is described in connection with an embodiment, it should be understood that, whether explicitly described or not, it is within the knowledge of those skilled in the art to affect such feature, structure, or characteristic in conjunction with other embodiments.

[0173] In the preparation methods described herein, steps may be performed in any order without departing from the principles of the invention, except where the timing or order of operations is explicitly stated. A statement in a claim that implies performing a step first, followed by several other steps, should be interpreted as meaning that the first step is performed before any other steps, but the other steps may be performed in any suitable order unless the order is further stated in the other steps. For example, a claim element stating "step A, step B, step C, step D, and step E" should be interpreted as meaning that step A is performed first, and step E is performed last, and steps B, C, and D may be performed in any order between steps A and E, and such order still falls within the literal scope of the claimed process. A given step or a subset of steps may also be repeated. Furthermore, unless the steps specified in the explicit claim language are performed individually, the specified steps may be performed simultaneously.

[0174] Other definitions of the selected terms used herein can be found in the detailed description of the invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0175] Industrial applicability:

[0176] This invention discloses that viral proteases include EV71 2A. pro and SARS-CoV-2 3CL pro It can enhance the extracellular concentration of LRPAP1, leading to IFNAR1 degradation and further inhibiting interferon signaling. Furthermore, inhibiting LRPAP1 with α2M can effectively reduce the infection rate of both RNA and DNA viruses. With the assistance of appropriate delivery systems, α2M holds promise as a broad-spectrum antiviral agent for clinical application.

[0177] This achievement not only deepens our understanding of the interaction mechanism between lipoprotein metabolism and host innate immunity, but also lays the foundation for developing LRPAP1 inhibitors as highly effective antiviral drugs.

[0178] References: The following references are incorporated herein by reference: 1 Wu, Y. et al., Main protease of SARS-CoV-2 serves as a bifunctional molecule in restricting type I interferon antiviral signaling. Signal Transduction and Targeted Therapy 5, 221, doi: 10.1038 / s41392-020-00332-2 (2020). 2 Lei, J. & Hilgenfeld, R. RNA-virus proteases counteracting host innate immunity. FEBS Lett 591, 3190-3210, doi: 10.1002 / 1873-3468.12827 (2017). 3 Matthews, K. A., Pham, A. & Frieman, M. The SARS coronavirus papain-like protease can inhibit IRF3 at a post-activation step that requires deubiquitination activity. Virology Journal 11, 209, doi: 10.1186 / s12985-014-0209-9 (2014). 4 Yang, H. & Rao, Z. Structural biology of SARS-CoV-2 and implications for therapeutic development. Nature Reviews Microbiology 19, 685-700, doi: 10.1038 / s41579-021-00630-8 (2021). 5 Sheng, Z., Prorok, M., Brown, BE & Castellino, FJ. Inhibition of N-methyl-D-aspartate receptor by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein. Neuropharmacology 55, 204-214, doi: 10.1016 / j.neuropharm.2008.05.016 (2008). 6 Mantuano, E. et al., LDL receptor-related protein-1 regulates NF-κB signaling pathway and microRNA-155 in macrophages to control the inflammatory response. Proc Natl Acad Sci USA 113, 1369-1374, doi: 10.1073 / pnas.1515480113 (2016). 7 Galliano, MF et al., Binding of alpha2ML1 to the low density lipoprotein receptor-related protein 1 (LRP1) reveals a new role for LRP1 in the human epidermis. PLoS One 3, e2729, doi: 10.1371 / journal.pone.0002729 (2008). 8 Willnow, T., Goldstein, J., Orth, K., Brown, M. & Herz, J. Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance. The Journal of Biological Chemistry 267, 26172-26180, doi: 10.1016 / S0021-9258(18)35732-6 (1993). 9 Willnow, TE, Sheng, Z., Ishibashi, S. & Herz, J. Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist. Science 264, 1471, doi: 10.1126 / science.7515194 (1994). 10 Wang, S. et al. identified α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis. Arthritis Rheumatol 66, 1843-1853, doi:10.1002 / art.38576 (2014). 11 Kanekiyo, T. & Bu, G. Receptor-associated protein interacts with amyloid-β peptide and promotes its cellular uptake. Journal of Biological Chemistry 284, 33352-33359 (2009). 12 Cater, JH, Wilson, MR & Wyatt, ARα2-Macroglobulin: a hypochlorite-regulated molecular chaperone and immune system modulator. Oxid Med Cell Longev 2019, 5410657, doi: 10.1155 / 2019 / 5410657 (2019). 13 Zhu, M., Zhao, B., Wei, L & Wang, S. alpha-2-Macroglobulin, a native and powerful proteinase inhibitor, prevents cartilage degeneration disease by inhibiting the majority of catabolic enzymes and cytokines. Current Molecular Biology Reports 7, 1-7, doi: 10.1007 / s40610-020-00142-z (2021).

Claims

1. A method for preventing and / or treating viral infections, characterized in that, The method involves administering an effective dose of a low-density lipoprotein receptor-associated protein 1 (LRPAP1) inhibitor to a subject in need, thereby increasing the expression levels of interferon and its derivatives in the subject's body.

2. The method of claim 1, wherein the viral infection includes diseases caused by DNA viruses or RNA viruses.

3. The method according to claim 2, wherein the DNA virus includes herpesvirus or hepatitis virus.

4. The method according to claim 2, wherein the RNA virus includes a small RNA virus, a coronavirus, an enterovirus, or a flavivirus.

5. The method of claim 1, wherein the LRPAP1 inhibitor comprises a pharmaceutical composition comprising α2-macroglobulin, a siRNA sequence having sequence number SEQ ID NO.: 1 or SEQ ID NO.: 2, a lysosomal inhibitor, or a combination thereof.

6. The method of claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

7. The method of claim 5, wherein the lysosomal inhibitor comprises methylamine or baffirizine.

8. The method according to claim 1, wherein the effective dose is within the biosafety range and does not exceed 1.5 mg / 100 mL.

9. The method according to claim 1, wherein the LRPAP1 inhibitor blocks the binding of the N-terminus of secreted LRPAP1 to the extracellular domain of interferon α / β receptor 1 (IFNAR1).

10. The method of claim 1, wherein the LRPAP1 inhibitor downregulates VP1 expression.

11. The method of claim 1, wherein the subject is further administered an IFNAR1 agonist, and the IFNAR1 agonist is administered before, after, or simultaneously with the administration of the LRPAP1 inhibitor.

12. The method of claim 10, wherein the concentration range of the IFNAR1 agonist is 0.01 to 10 mg / mL.

13. The method according to claim 1, wherein the interferon and its derivatives include natural / recombinant interferon, conformationally modified interferon, interferon conjugates, or interferon fusion proteins.

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