Application based on EBNA1 protein GR2 region of EB virus mediating liquid-liquid phase separation function

Abstract

The application discloses application of a GR2 region of an EB virus EBNA1 protein mediating liquid-liquid phase separation function, relates to the field of biotechnology, and provides a screening method for a medicine for preventing and / or treating EB virus infection or an EB virus related disease, and comprises the following steps: taking the GR2 region of the EBNA1 as a target, screening an active substance for inhibiting the GR2 region from mediating liquid-liquid phase separation, and then obtaining the medicine; the sequence of the EBNA1 is shown as SEQ ID NO:1; and the sequence of the GR2 region is shown as SEQ ID NO:2. The application finds that the GR2 region of the EBNA1 is a key functional region for mediating liquid-liquid phase separation, and the liquid-liquid phase separation process is indispensable for stable maintenance of an EB virus genome. Therefore, the required medicine can be obtained by screening the active substance for inhibiting the GR2 region from mediating liquid-liquid phase separation of the EBNA1.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of liquid-liquid phase separation mediated by the GR2 region of the EBNA1 protein of the Epstein-Barr virus. Background Technology

[0002] Liquid-liquid phase separation (LLPS) is an important biophysical process within cells. Through multivalent interactions mediated by intrinsically disordered regions (IDRs) in biomolecules such as proteins, it condenses specific proteins and nucleic acids into membrane-free compartments, forming condensates. These condensates provide a relatively stable microenvironment for a range of biological reactions and play a crucial role in various cellular functions. In recent years, LLPS has attracted significant attention in the field of virology. Studies have found that many viruses can utilize the host's LLPS mechanism or the phase-separation ability of viral proteins themselves to construct viral replication compartments, thereby efficiently recruiting viral and host factors to promote viral genome replication, maintenance, and gene expression. For example, the latent-associated nuclear antigen (LANA) protein of Kaposi's sarcoma-associated herpesvirus (KSHV) can form nucleosomes (LANA-NBs) through liquid-liquid phase separation, which is crucial for the replication and stability of the KSHV genome; the E2 protein of human papillomavirus type 16 (HPV16) can also form liquid-liquid phase separation through interaction with the host p53 protein to assist viral replication.

[0003] Epstein-Barr virus (EBV) is a gamma herpesvirus that widely infects humans and is closely associated with the development of various diseases. During the latent infection period, the EBV genome persists in the host's B lymphocytes in the form of a circular exosome, and its stable maintenance is the cornerstone of latent infection and related oncogenic mechanisms. EBV nuclear antigen 1 (EBNA1) is a viral protein that is continuously expressed throughout the latent infection phase and is indispensable for the replication, distribution, and long-term maintenance of the viral exosome. Therefore, it can serve as a core target for intervening in latent EBV infection and treating related diseases. However, current understanding of the mechanism of action of EBNA1 is mainly limited to its dimerization and DNA-binding characteristics, failing to reveal the possible liquid-liquid phase separation driving mechanism at the subcellular spatial tissue level, similar to other viral proteins. Existing strategies against latent EBV infection lack intervention methods targeting the physical process of EBNA1 phase separation. This technological gap limits researchers' ability to develop novel intervention strategies targeting EBNA1.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an application based on the liquid-liquid phase separation function mediated by the GR2 region of the EBNA1 protein of EB virus, which aims to provide a new therapeutic target for EB virus infection and further for screening drugs to treat EB virus infection.

[0006] The technical solution of the present invention is as follows: A first aspect of the present invention provides a method for screening drugs for the prevention and / or treatment of EB virus infection or EB virus-related diseases, comprising the following steps: Targeting the GR2 region of EBNA1, we screen for active substances that inhibit the GR2 region-mediated liquid-liquid phase separation of EBNA1, thereby obtaining drugs for the prevention and / or treatment of EB virus infection or EB virus-related diseases. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO: 2.

[0007] A second aspect of the present invention provides the use of an active substance in the preparation of a medicament for the prevention and / or treatment of EB virus infection or EB virus-related diseases, said active substance inhibiting EBNA1 liquid-liquid phase separation mediated by the GR2 region of EBNA1; The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO: 2.

[0008] A third aspect of the present invention provides a method for regulating the ability and function of EBNA1 in liquid-liquid phase separation, comprising the following steps: The ability and function of EBNA1 to separate liquid phases can be regulated by deleting the GR2 region of EBNA1 or by mutating the arginine in amino acids 353-379 of the GR2 region of EBNA1. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO: 2.

[0009] Optionally, all arginine amino acids at positions 353-379 in the GR2 region of EBNA1 are mutated to alanine.

[0010] In a fourth aspect, the present invention provides an EBNA1 mutant, wherein the EBNA1 mutant is obtained by deleting or mutating the GR2 region of EBNA1. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1, and the amino acid sequence of the GR2 region is shown in SEQ ID NO: 2. Specifically, the GR2 region is mutated by mutating the arginine in amino acids 353-379 of the GR2 region.

[0011] Optionally, the GR2 region can be mutated by replacing all arginine amino acids at positions 353-379 of the GR2 region with alanine.

[0012] In a fifth aspect, the present invention provides a nucleic acid, wherein the nucleic acid encodes the EBNA1 mutant of the present invention as described above.

[0013] In a sixth aspect, the present invention provides an expression vector containing the nucleic acid of the present invention as described above.

[0014] A seventh aspect of the present invention provides a cell containing an expression vector of the present invention as described above.

[0015] An eighth aspect of the present invention provides a kit comprising at least one of the following substances (1) to (4): (1) The EBNA1 mutant of the present invention as described above; (2) The nucleic acid of the present invention as described above; (3) The expression vector of the present invention as described above; (4) The cell of the present invention as described above.

[0016] Beneficial Effects: This invention reveals that EBNA1 possesses an intrinsic ability to form liquid-liquid phase separation condensates, which can form fusion-like, dynamically exchangeable, and 1,6-hexanediol-sensitive biomolecular condensates within the cell nucleus. Furthermore, it clarifies that the GR2 region is a key functional region mediating EBNA1 liquid-liquid phase separation, and that this process is indispensable for the stable maintenance of the EBV genome. Therefore, this invention provides a novel target for the prevention and / or treatment of EBV infection or EBV-related diseases—the GR2 region of EBNA1. By screening for active substances that inhibit the GR2 region-mediated EBNA1 liquid-liquid phase separation, drugs for the prevention and / or treatment of EBV infection or EBV-related diseases can be obtained.

[0017] Furthermore, based on the discovery that the GR2 region is a key functional region mediating EBNA1 liquid-liquid phase separation, this invention further enables the regulation of the ability and function of EBNA1 liquid-liquid phase separation by deleting or mutating the GR2 region of EBNA1; and further provides EBNA1 mutants and their encoding nucleic acids, expression vectors, cells containing the expression vectors, and related kits that can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or intervene in the maintenance of EB virus latency. Attached Figure Description

[0018] Figure 1 The images show the results of EBNA1's mediated liquid-liquid phase separation in cells. A shows the results of predictive analysis of the intrinsic disordered regions in the EBNA1 amino acid sequence using the IUPred2 algorithm; B shows the results of fluorescence microscopy confirming EBNA1's ability to form intranuclear condensates within cells; C shows the results of live-cell time-series observation of HEK293T cells expressing the EGFP-EBNA1 fusion protein; D shows the results of a fluorescence bleaching recovery experiment on intranuclear condensates formed by the EGFP-EBNA1 fusion protein; and E shows fluorescence microscopy images of HEK293T cells expressing the EGFP-EBNA1 fusion protein with and without 1,6-hexanediol treatment.

[0019] Figure 2 The diagrams show the results of liquid-liquid phase separation mediated by the GR2 domain. A shows the fluorescence observation results of HEK293T cells transfected with an expression vector containing the EGFP-labeled EBNA1 truncated mutant ΔGR2. B shows the fluorescence observation results after transfecting HEK293T cells with EGFP-labeled GR1 and EGFP-labeled GR2 expression vectors, respectively. C shows the fluorescence observation results of HEK293T cells expressing EGFP-labeled GR2 with or without 1,6-hexanediol treatment. D shows the recovery results of nuclear spots formed by EGFP-labeled GR2 after fluorescence bleaching. E shows the amino acid sequence diagrams of the three GR2 region mutants. F shows the subcellular localization results of the EGFP-labeled GR2 region mutant overexpressed in HEK293T cells.

[0020] Figure 3 The images show the results of EBV genome copy number detection after liquid-liquid phase separation and infection. In the images, A shows the EBV genome copy number results after Akata cells were treated with or without 1,6-hexanediol, and B shows the EBV genome copy number results after Akata cells were transfected with different expression vectors. Detailed Implementation

[0021] This invention provides an application based on the liquid-liquid phase separation function mediated by the GR2 region of the EBNA1 protein of the Epstein-Barr virus. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0024] This invention discovers that the GR2 region (amino acids 325-379) of EBNA1 (Epstein-Barr virus nuclear antigen 1), rich in glycine (Gly) and arginine (Arg), is a key region regulating its liquid-liquid phase separation ability and is an important condition for the maintenance of EB virus latency. Based on this discovery, this invention provides the following applications of liquid-liquid phase separation function mediated by the GR2 region of the EBNA1 protein.

[0025] This invention provides a method for screening drugs for the prevention and / or treatment of EBV infection or EBV-related diseases, comprising the following steps: Targeting the GR2 region of EBNA1, we screen for active substances that inhibit the GR2 region-mediated liquid-liquid phase separation of EBNA1, thereby obtaining drugs for the prevention and / or treatment of EB virus infection or EB virus-related diseases (including active substances that inhibit the GR2 region-mediated liquid-liquid phase separation of EBNA1). The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO: 2 (positions 325-379 of the EBNA1 amino acid sequence).

[0026] This invention, through experimental research, has discovered that EBNA1 undergoes liquid-liquid phase separation through its specific intrinsic disordered region G2, forming dynamic intranuclear biomolecule condensates. This physicochemical process plays an indispensable role in maintaining the stability of the EB virus genome, thus providing a novel mechanism of action, experimental evidence, and therapeutic target for targeting and interfering with EBNA1 phase separation to treat EB virus-related diseases.

[0027] Specifically, EBNA1 is indispensable for the replication, distribution, and long-term maintenance of viral isolates. This invention reveals that liquid-liquid phase separation of EBNA1 plays a crucial role in maintaining latent EBV infection. The intrinsic disordered region of EBNA1 (specifically the GR2 region) is a key structural domain driving liquid-liquid phase separation and the formation of aggregates. Therefore, by screening for active substances that inhibit GR2 region-mediated EBNA1 liquid-liquid phase separation, drugs for the prevention and / or treatment of EBV infection or EBV-related diseases can be obtained.

[0028] Specifically, active substances that inhibit GR2 region-mediated EBNA1 liquid-liquid phase separation can be screened by detecting the effect of the analyte on the formation, stability, or dynamic characteristics of GR2 region-mediated liquid-liquid phase separation.

[0029] In some embodiments, the active substance that inhibits the GR2 region-mediated liquid-liquid phase separation of EBNA1 can be a small molecule compound, a protein or its functional fragment, an antibody or antigen-binding fragment, a peptide, a peptide-like substance, or a nucleic acid molecule, etc.

[0030] This invention also provides the use of an active substance in the preparation of a medicament for the prevention and / or treatment of EBV infection or EBV-related diseases, wherein the active substance inhibits EBNA1 liquid-liquid phase separation mediated by the GR2 region of EBNA1; The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO: 2.

[0031] EBNA1 is essential for the replication, distribution, and long-term maintenance of viral isolates. This invention reveals that liquid-liquid phase separation of EBNA1 plays a crucial role in maintaining latent EBV infection. Specifically, the intrinsic disordered region of EBNA1 (the GR2 region) is a key structural domain driving liquid-liquid phase separation and the formation of aggregates. Therefore, active substances capable of inhibiting the GR2 region-mediated liquid-liquid phase separation of EBNA1 can be used for the prevention and / or treatment of EBV infection or EBV-related diseases.

[0032] In some specific embodiments, the active substance may be a small molecule compound, a protein or its functional fragment, an antibody or antigen-binding fragment, a peptide, a peptide-like substance, or a nucleic acid molecule, etc.

[0033] This invention also provides a method for regulating the liquid-liquid phase separation capability and function of EBNA1, comprising the following steps: The ability and function of EBNA1 to separate liquid phases can be regulated by deleting the GR2 region of EBNA1 or by mutating the arginine in amino acids 353-379 of the GR2 region of EBNA1. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO: 2.

[0034] This invention discovers that the intrinsic disordered region of EBNA1 (specifically the GR2 region) is the key structural domain driving EBNA1 to undergo liquid-liquid phase separation and generate condensates. Furthermore, by deleting the GR2 region of EBNA1 or mutating the arginine in amino acids 353-379 of the GR2 region of EBNA1, the ability and function of EBNA1 to undergo liquid-liquid phase separation can be regulated (reduced, inhibited, weakened, or destroyed).

[0035] In some implementations, inhibition of EBNA1 liquid-liquid phase separation is achieved in cells and in vitro models by deleting the GR2 region of EBNA1 or by mutating all arginine amino acids at positions 353-379 of the GR2 region of EBNA1 to alanine.

[0036] Specifically, the GR2 region of EBNA1 is deleted or all arginine amino acids in the GR2 region of EBNA1 from position 353 to 379 are mutated to alanine to form the EBNA1 mutant; then the expression vector of this EBNA1 mutant is constructed and this expression vector is transferred into cells or in vitro models to inhibit the liquid-liquid phase separation of EBNA1.

[0037] This invention also provides an EBNA1 mutant, wherein the EBNA1 mutant is obtained by deleting or mutating the GR2 region of EBNA1 (i.e., wild-type EBNA1); The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1, and the amino acid sequence of the GR2 region is shown in SEQ ID NO: 2. The specific mutation of the GR2 region is to mutate the arginine in the amino acids at positions 353-379 of the GR2 region.

[0038] Specifically, the GR2 region of EBNA1 was deleted, that is, amino acids 325-379 were deleted from EBNA1. The amino acid sequence of the resulting EBNA1 mutant is shown in SEQ ID NO: 3.

[0039] Compared to the wild-type EBNA1 with an amino acid sequence as shown in SEQ ID NO: 1, the EBNA1 mutant provided by this invention has reduced or no liquid-liquid phase separation ability, which can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or as a tool to intervene in the maintenance of EB virus latency (by introducing the EBNA1 mutant into the cell, interfering with EBNA1 liquid-liquid phase separation and disrupting the maintenance of EB virus latency).

[0040] In some implementations, the GR2 region is mutated by replacing all arginine amino acids at positions 353-379 of the GR2 region with alanine.

[0041] This invention also provides a nucleic acid, wherein the nucleic acid encodes the EBNA1 mutant described above.

[0042] The nucleic acid provided in this embodiment can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or as a tool to intervene in the maintenance of EB virus latency.

[0043] This invention also provides an expression vector, wherein the expression vector contains the nucleic acid described above.

[0044] In some embodiments, the nucleotide sequence of the expression vector is shown in SEQ ID NO: 4.

[0045] The expression vector provided in this embodiment can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or as a tool to intervene in the maintenance of EB virus latency.

[0046] This invention also provides a cell containing the expression vector of this invention as described above.

[0047] The cells provided in this embodiment can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or serve as a tool for intervening in EB virus latency maintenance.

[0048] This invention also provides a kit containing at least one of the following substances (1) to (4): (1) The EBNA1 mutant described above in the embodiments of the present invention; (2) The nucleic acid described in the embodiments of the present invention; (3) The expression vector described above in the embodiments of the present invention; (4) The cells described above in the embodiments of the present invention.

[0049] The kit provided in this embodiment can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or as a tool to intervene in EB virus latency maintenance.

[0050] The present invention will be further described below through specific embodiments.

[0051] Example 1: Study on the formation of EBNA1 in cell-mediated liquid-liquid phase separation (1) Research on the structural basis of EBNA1 for forming liquid-liquid phase separation The UPred2 algorithm was used to predict and analyze the intrinsic disordered regions in the amino acid sequence of the EBNA1 protein (amino acid sequence shown in SEQ ID NO: 1, its NCBI gene number is Gene ID: 3783709). The results are as follows: Figure 1 As shown in A (where the predicted disordered regions are marked with black stripes), the results show that its sequence contains multiple possible intrinsically disordered regions (GR1, GA, GR2 and DBD), which provides a theoretical structural basis for its multivalent interactions and driving liquid-liquid phase separation.

[0052] (2) Research on the ability of EBNA1 to form intranuclear condensates within cells An expression vector labeled with EGFP (enhanced green fluorescent protein) was transfected into HEK293T cells (i.e., HEK293T cells expressing the EGFP-EBNA1 fusion protein). HEK293T cells transfected with an empty EGFP vector were used as a negative control. Immunofluorescence imaging results are as follows: Figure 1 As shown in Figure B, EGFP expressed by HEK293T cells after transfection with the empty EGFP vector is mainly diffusely distributed. After HEK293T cells are transfected with the expression vector of EGFP-labeled EBNA1, the expressed EGFP-EBNA1 fusion protein forms obvious punctate and aggregate-like fluorescent signals in the cell nucleus. In other words, the EGFP-EBNA1 fusion protein forms a large number of significant and discrete punctate aggregates in the cell nucleus, indicating that EBNA1 has the ability to form intranuclear aggregates in the cell. This preliminarily confirms that EBNA1 has the tendency to self-assemble into specific structures in the living cell environment.

[0053] (3) Study on the liquid-liquid phase separation characteristics of EBNA1 condensates To confirm that the condensate possessed typical characteristics of liquid-liquid phase separation, its dynamic behavior was further observed. Specifically, live-cell time-series observations were performed on HEK293T cells expressing the EGFP-EBNA1 fusion protein, and the results are as follows: Figure 1As shown in C, the point-like structures formed by EBNA1 in the nucleus can come into contact and fuse with each other, exhibiting typical droplet-like dynamic behavior.

[0054] (4) Study on the dynamic characteristics of EBNA1 condensates conforming to liquid-liquid phase separation. Next, fluorescence recovery after bleaching (FRAP) assays were performed on the nuclear condensates formed by the EGFP-EBNA1 fusion protein to quantitatively analyze their dynamic properties. The results are as follows: Figure 1 As shown in D, the fluorescence signal of the bleached area can be recovered in a short time, which proves that the EBNA1 protein molecules in the aggregate have high mobility, thus proving that EBNA1 forms a dynamic, liquid phase-separated aggregate in the cell, that is, the aggregate formed by EBNA1 conforms to the dynamic characteristics of liquid-liquid phase separation.

[0055] (5) Research on the core structure of EBNA1 belonging to liquid-liquid phase separation condensate. HEK293T cells expressing the EGFP-EBNA1 fusion protein were treated for 10 minutes in DMEM medium with or without 5% 1,6-hexanediol, and then counterstained with DAPI (4',6-diamidindo-2-phenylindole) for fluorescence observation. The results are as follows: Figure 1 As shown in E, the results show that after treatment with 1,6-hexanediol, the nuclear condensates formed by the EGFP-EBNA1 fusion protein were significantly reduced or dissipated, indicating that the condensates are sensitive to the liquid-liquid phase separation disruptor (1,6-hexanediol). In other words, the nuclear structure formed by EBNA1 belongs to the liquid-liquid phase separation condensate.

[0056] Furthermore, the present invention validated the liquid-liquid phase separation capability of EBNA1 in Akata and Raji cells, which is consistent with the above conclusions.

[0057] In summary, this embodiment reveals that EBNA1 can undergo liquid-liquid phase separation and form dynamic intranuclear condensates. Existing research on the function of EBNA1 mainly focuses on its role in binding to the Epstein-Barr virus (EBV) genome replication origin OriP through its DNA-binding domain, mediating viral episome replication and distribution, and this understanding is primarily based on a static protein-DNA binding model. This embodiment, for the first time, reveals from the perspective of biomolecular phase behavior that EBNA1 possesses the intrinsic ability to form liquid-liquid phase-separated condensates, capable of forming fusion-like, dynamically exchangeable, and 1,6-hexanediol-sensitive biomolecular condensates within the cell nucleus. This discovery indicates that, in addition to its traditional DNA-binding function, EBNA1 can also participate in viral genome maintenance-related biological processes by forming dynamic functional compartments through phase separation.

[0058] Example 2: Study on the GR2 region as a key region mediating liquid-liquid phase separation (1) Research and verification of the key role of the GR2 region in the formation of EBNA1-mediated intranuclear condensates A specific truncated mutant (ΔGR2) for EGFP fusion expression was constructed using bridging PCR technology targeting the predicted disordered region, and its expression vector was constructed, including the following steps: Four specific primers were designed and synthesized for bridging PCR targeting the wild-type EBNA1 gene coding sequence (Gene ID: 3783709). These were ΔGR2-Fw, starting upstream of the start site of the GR2 domain to be deleted (its amino acid sequence is shown in SEQ ID NO: 2); ΔGR2-Rv, starting downstream of the GR2 domain termination site; and primers EBNA1-Fw and EBNA1-Rv, located upstream and downstream of wild-type EBNA1 and containing NotI and BamHI restriction sites, respectively. Primers ΔGR2-Fw and EBNA1-Fw, and ΔGR2-Rv and EBNA1-Rv, were amplified using wild-type EBNA1 as a template, digested with TaqI, and ligated. After purification of the enzyme ligation product, it was used as a template for amplification with upstream and downstream primers of EBNA1. The amplified product was digested with NotI and BamHI and then ligated into the EGFP-C1 vector to obtain the first expression vector (which expresses the fusion protein of EGFP and the EBNA1 truncated mutant △GR2). The nucleotide sequence of the first expression vector is shown in SEQ ID NO: 4.

[0059] The specific information for each primer is shown in Table 1.

[0060] Table 1. Primer Information

[0061] The first expression vector was transfected into HEK293T cells (i.e., HEK293T cells expressing the fusion protein of EGFP and the EBNA1 truncated mutant ΔGR2), and fluorescence observation was performed. The results are as follows: Figure 2 As shown in A, the results indicate that the EBNA1 truncation mutant △GR2 completely loses its ability to form nuclear condensates (the ability to form nuclear spots is significantly reduced or disappears).

[0062] The above results indicate that the GR2 region plays a crucial role in EBNA1-mediated formation of intranuclear condensates.

[0063] (2) Research and verification of the ability of the GR2 region itself to independently drive the formation of aggregates. The expression vectors of EGFP-labeled GR1 and EGFP-labeled GR2 were transfected into HEK293T cells, and fluorescence was observed. The results are as follows: Figure 2As shown in Figure B, the results indicate that EGFP-labeled GR2 can form distinct dot-like aggregates within the cell nucleus, while EGFP-labeled GR1 does not form similar distinct intranuclear spots. In other words, when the GR2 domain is fused with EGFP protein alone, the EGFP-GR2 fusion protein still exhibits liquid-liquid phase separation in cells. This result suggests that the GR2 region itself has the ability to independently drive aggregate formation.

[0064] (3) The aggregated structure formed by GR2 is sensitive to liquid-liquid phase separation disruptors and exhibits liquid-liquid phase separation characteristics. The EGFP-labeled GR2 expression vector was transfected into HEK293T cells. After treatment with DMEM medium (with or without 3.5% 1,6-hexanediol) for 10 minutes, fluorescence was observed, and the number of spots in each cell was statistically analyzed. The results are as follows: Figure 2 As shown in C, the results indicate that 1,6-hexanediol treatment significantly reduces the number of core spots formed by GR2. This result suggests that the aggregated structures formed by GR2 are sensitive to liquid-liquid phase separation disruptors (1,6-hexanediol) and exhibit liquid-liquid phase separation characteristics.

[0065] (4) Research and verification of the inherent driving liquid-liquid phase separation capability of the GR2 region The results of the fluorescence bleaching recovery assay were performed on the intranuclear spots (i.e., punctate aggregates) formed by EGFP-labeled GR2. Figure 2 As shown in Figure D, the results indicate that the fluorescence signal recovers rapidly after bleaching, suggesting that the GR2 aggregates exhibit dynamic and reversible molecular exchange characteristics. This result demonstrates that the GR2 region possesses an intrinsic ability to drive liquid-liquid phase separation.

[0066] like Figure 2 As shown in E, this embodiment constructed three GR2 region mutants: Based on the wild-type GR2 region (whose amino acid sequence is shown in SEQ ID NO: 2, located at positions 325-379 of the EBNA1 amino acid sequence shown in SEQ ID NO: 1), all arginine (R) at positions 325-347 are mutated to alanine (A) to obtain GR2-6.

[0067] Based on the wild-type GR2, all arginine (R) at positions 353-379 were mutated to alanine (A) to obtain GR2-12.

[0068] Based on the wild-type GR2 region, all arginine (R) was mutated to alanine (A) to obtain GR2-18.

[0069] EGFP-labeled GR2-6 expression vector, EGFP-labeled GR2-12 expression vector, and EGFP-labeled GR2-18 expression vector were transfected into HEK293T cells (i.e., EGFP-GR2-6 fusion protein, EGFP-GR2-12 fusion protein, and EGFP-GR2-18 fusion protein were expressed in HEK293T cells, respectively). Subcellular localization was then performed, and the results are as follows: Figure 2 As shown in F, the EGFP-GR2-6 fusion protein can form dot-like aggregates, while the EGFP-GR2-12 and EGFP-GR2-18 fusion proteins cannot. This indicates that by mutating all arginine residues (353-379) in the GR2 region to alanine, dot-like aggregates cannot be formed. The GR2 domain plays a crucial role in EBNA1-mediated liquid-liquid phase separation, thus localizing the core functional domain driving phase separation to this specific disordered region. The GR2 region mediates the liquid-liquid phase separation of EBNA1.

[0070] In summary, this embodiment clarifies that the GR2 region is a key functional region mediating liquid-liquid phase separation in EBNA1. Specifically, this embodiment further clarifies that the GR2 region (325-379 aa) in the EBNA1 protein is a key functional region mediating its liquid-liquid phase separation ability. Compared with wild-type EBNA1, the ability of EBNA1 to form intranuclear condensates is significantly reduced or disappeared after the GR2 region is missing. At the same time, the GR2 region itself can form condensates with liquid-liquid phase separation characteristics, indicating that the GR2 region is not only a necessary structural unit, but also a key region sufficient to drive phase separation formation. In particular, the ability of the GR2 region to form condensates itself is significantly lost after the arginine mutation at positions 353-379, indicating that arginine at positions 353-379 in the GR2 region plays an important role in the ability of GR2 to drive phase separation.

[0071] Example 3: Study on the relationship between GR2-mediated liquid-liquid phase separation and maintenance of latent EB virus infection. In this embodiment, a functional intervention experiment was conducted to establish a direct causal relationship between GR2-mediated phase separation capability and EBNA1 maintaining EB virus genome stability.

[0072] Specifically, to elucidate the function of GR2-mediated liquid-liquid phase separation in EBV genome replication and maintenance, cells were treated with 1,6-hexanediol, a reagent known to disrupt weak interactions. Specifically, Akata cells were treated with 0.75% 1,6-hexanediol for 6 hours, after which genomic DNA was extracted. EBV genome copy number was quantified by qPCR using EBNA1 as the detection target. Results... Figure 3As shown in A, the results showed that treatment of cells with 1,6-hexanediol significantly reduced the EB virus genome copy number, indicating that disruption of liquid-liquid phase separation interferes with the binding of the EB virus genome to the EBNA1 protein in the phase-separated droplets, impairing the maintenance and replication of the EBNA1-dependent viral genome.

[0073] Akata cells were transfected with different expression vectors: an expression vector for the EGFP-wild-type EBNA1 fusion protein and an expression vector for the EGFP-EBNA1 truncated mutant ΔGR2 fusion protein (i.e., the first expression vector, whose nucleotide sequence is shown in SEQ ID NO: 4). This resulted in overexpression of the EGFP-wild-type EBNA1 fusion protein and the aforementioned EGFP-EBNA1 truncated mutant ΔGR2 fusion protein, both of which lacked phase-separation ability, in Akata cells. Untransfected blank cells were used as a control. The intracellular EBV genome copy number was then detected by qPCR. The results are shown below. Figure 3 As shown in Figure B, the results indicate that overexpression of the EGFP-EBNA1 truncated mutant ΔGR2 fusion protein (i.e., EBNA1 ΔGR2 in the figure) reduces the viral genome copy number, suggesting that GR2-mediated liquid-liquid phase separation is crucial for maintaining EBV latency. Therefore, 1,6-hexanediol treatment of cells can introduce EBNA1 variants with lost GR2 function or reduced phase separation ability, interfering with multivalent interactions in the GR2 region, interfering with GR2-related post-translational modifications, or blocking the formation of functional intranuclear condensates by EBNA1, thereby reducing EBV genome stability or copy number.

[0074] In summary, this embodiment reveals the functional association between GR2-mediated liquid-liquid phase separation and EBNA1's role in maintaining viral genome stability. Specifically, this embodiment found that the GR2 region-mediated EBNA1 liquid-liquid phase separation capability is closely related to the stable maintenance of the EBV genome in latently infected cells. Disruption of the GR2 region, interference with phase separation, or treatment with phase separation perturbators reduced EBNA1-related aggregate formation ability, accompanied by a decrease in EBV genome copy number or impaired viral genome stability. These results indicate that GR2-mediated liquid-liquid phase separation is a crucial mechanistic basis for EBNA1's latent maintenance function, thus providing a novel mechanism, experimental evidence, and precise molecular target for targeting interference with EBNA1 phase separation to treat EBV infection or EBV-related diseases.

[0075] Applications that can be made based on the results of the above embodiments: Targeting the GR2 region of EBNA1, this study aims to screen for active substances that inhibit GR2-mediated liquid-liquid phase separation of EBNA1, thereby obtaining drugs for the prevention and / or treatment of EBV infection or EBV-related diseases. These active substances weaken the ability of EBNA1 to form functional intranuclear condensates by interfering with the multivalent interactions and liquid-liquid phase separation mediated by the GR2 region, thus affecting the maintenance of EBV latency.

[0076] Active substances that inhibit GR2 region-mediated EBNA1 liquid-liquid phase separation can be used to prevent and / or treat EB virus infection or EB virus-related diseases.

[0077] The ability and function of EBNA1 protein to separate liquid phases can be regulated by deleting the GR2 region of EBNA1 or by mutating all arginine amino acids at positions 353-379 of the GR2 region of EBNA1 to alanine.

[0078] EBNA1 mutants obtained by deleting or mutating the GR2 region of EBNA1, their encoding nucleic acids, expression vectors, cells containing the expression vectors, and kits containing at least one of the above substances can be used to study the EBNA1 phase separation mechanism, evaluate the EB virus genome maintenance function, or serve as a tool for intervening in EB virus latency maintenance.

[0079] In summary, this invention reveals and confirms that EBNA1 forms dynamic intranuclear condensates through a liquid-liquid phase separation mechanism, which is crucial for maintaining viral genome stability. It also elucidates that its specific intrinsically disordered GR2 region is the core structural domain driving phase separation, and demonstrates that disrupting this liquid-liquid phase separation process can affect the stability of the EB virus genome. Therefore, this provides a direct mechanistic basis and precise molecular target for developing novel therapeutic strategies to clear latent EB virus infection by targeting GR2-mediated EBNA1 phase separation. Specifically: (1) Bioinformatics prediction combined with cell imaging verification: Compared with single experimental speculation, this invention realizes the connection from theoretical structure prediction to cell phenotype observation. Through software prediction, it was found that EBNA1 contains an internal disordered region, which provides a theoretical basis for its phase separation potential. Furthermore, through fluorescent fusion protein expression and imaging, it was directly confirmed that EBNA1 can form specific punctate aggregates in the cell nucleus, providing a solid morphological starting point for subsequent in-depth research on phase separation properties.

[0080] (2) Dynamic fusion observation and fluorescence bleaching recovery analysis: By monitoring the fusion and splitting dynamics of the condensate and combining it with fluorescence bleaching recovery technology for quantitative analysis, the physical properties of the subcellular structure were accurately characterized. This directly verified that the molecules inside the EBNA1 condensate have the ability to diffuse and exchange rapidly, thus clearly defining it as a phase-separated structure with dynamic liquid properties rather than a static solid aggregate, laying a key physicochemical foundation for subsequent functional studies.

[0081] (3) Construction and characterization of key domain truncation mutants: By constructing and expressing specific deletion mutants, the genetic manipulation of EBNA1 phase separation ability is realized, and the core function driving phase separation is precisely locked to the specific disordered GR2 region. This realizes the causal relationship from phenotype to the decisive domain, providing a precise molecular target for designing targeted inhibitors.

[0082] (4) Chemical perturbation and genetic function recovery experiment: By using a phase separation disruptor (1,6-hexanediol) for chemical disruption and long-term genetic intervention with expression loss mutants, a two-way functional verification model was constructed. The results jointly confirmed that the disruption of GR2-mediated EBNA1 phase separation directly led to the impairment of viral genome maintenance function, thus establishing a causal relationship between protein phase separation ability and the core biological function of maintaining viral genome stability, proving the effectiveness of targeting this process.

[0083] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for screening drugs for the prevention and / or treatment of EBV infection or EBV-related diseases, characterized in that, Includes the following steps: Targeting the GR2 region of EBNA1, we screen for active substances that inhibit the GR2 region-mediated liquid-liquid phase separation of EBNA1, thereby obtaining drugs for the prevention and / or treatment of EB virus infection or EB virus-related diseases. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO:

2.

2. The use of an active substance in the preparation of a medicament for the prevention and / or treatment of EBV infection or EBV-related diseases, wherein the active substance inhibits EBNA1 liquid-liquid phase separation mediated by the GR2 region of EBNA1; The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO:

2.

3. A method for regulating the liquid-liquid phase separation capability and function of EBNA1, characterized in that, Includes the following steps: The ability and function of EBNA1 to separate liquid phases can be regulated by deleting the GR2 region of EBNA1 or by mutating the arginine in amino acids 353-379 of the GR2 region of EBNA1. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1; The amino acid sequence of the GR2 region is shown in SEQ ID NO:

2.

4. The method according to claim 3, characterized in that, All arginine amino acids in the GR2 region of EBNA1 from position 353 to 379 were mutated to alanine.

5. An EBNA1 mutant, characterized in that, The EBNA1 mutant was obtained by deleting or mutating the GR2 region of EBNA1. The amino acid sequence of EBNA1 is shown in SEQ ID NO: 1, and the amino acid sequence of the GR2 region is shown in SEQ ID NO:

2. Specifically, the GR2 region is mutated by mutating the arginine in amino acids 353-379 of the GR2 region.

6. The EBNA1 mutant according to claim 5, characterized in that, Specifically, the GR2 region was mutated by replacing all arginine amino acids at positions 353-379 of the GR2 region with alanine.

7. A nucleic acid, characterized in that, Encodes the EBNA1 mutant as described in any one of claims 5-6.

8. An expression carrier, characterized in that, It contains the nucleic acid as described in claim 7.

9. A cell characterized by, It contains the expression vector as described in claim 8.

10. A reagent kit, characterized in that, Contains at least one of the following substances (1) to (4): (1) The EBNA1 mutant according to any one of claims 5-6; (2) The nucleic acid as described in claim 7; (3) The expression vector according to claim 8; (4) The cell according to claim 9.

Images