Use of tiar inhibitors for the treatment of hepatitis b virus infection

Abstract

The present application discloses the use of TIAR inhibitors for treating hepatitis B virus infection. The present application demonstrates that the host factor TIAR promotes HBV replication by regulating the translation of pgRNA through its binding, i.e. inhibiting the translation of nucleocapsid protein but promoting the translation of Pol. It is also found that TIAR is also packaged into the nucleocapsid of HBV through its binding to pgRNA. The present application provides a new solution for treating HBV infection based on TIAR as a target by reducing the expression of TIAR or inhibiting the binding of TIAR to pgRNA to inhibit HBV replication.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hepatitis B virus therapy, in particular to the use of TIAR inhibitors for treating hepatitis B virus infection, methods for modulating hepatitis B virus activity and methods for screening substances useful for treating hepatitis B virus infection. BACKGROUND

[0002] Currently, the drugs clinically used for treating hepatitis B virus (HBV) infection mainly include nucleos(t)ide analogues (NAs) and interferon-alpha (IFN-α), both of which can efficiently inhibit viral replication, but none can effectively cure hepatitis B.

[0003] HBV belongs to the Orthohepadnavirus genus of the Hepadnaviridae family, and its genome is a 3.2 kb long partially double-stranded, relaxed circular DNA (rcDNA). In the HBV life cycle, HBV first binds to the sodium taurocholate co-transporting polypeptide (NTCP) receptor on the membrane of hepatocytes through the amino-terminal domain of its large envelope protein PreS1, and selectively enters human hepatocytes. The rcDNA released from the HBV nucleocapsid enters the nucleus of the hepatocyte and is repaired to form a covalently closed circular DNA (cccDNA). In the nucleus of infected hepatocytes, the cccDNA exists in the form of a mini-chromosome and can transcribe various viral RNAs, among which the 3.5 kb pregenomic RNA (pgRNA) plays an extremely important role in the HBV replication cycle. The pgRNA is both a template for reverse transcription to form rcDNA and a bi-cistronic mRNA for translation to produce core and polymerase (Pol) proteins. Once formed, the Pol protein can recognize and bind to the core packaging signal-5' end epsilon stem loop structure on the pgRNA, and then initiate the assembly of the viral capsid and the reverse transcription of the daughter virus rcDNA.

[0004] In addition, both HBV infection and replication depend on the regulation of various host factors in hepatocytes. Among them, T-cell intracellular antigen 1-related protein (TIAR) is a key protein of the eukaryotic classic stress granule (SG). When eukaryotic cells are stimulated by external environment such as viral infection, the phosphorylation of the 51st serine of the eIF2α translation initiation factor occurs, resulting in the blockage of mRNA translation. These translation-blocked mRNAs can form dense, membrane-unwrapped granular aggregates with TIAR, translation initiation factors, and RNA-binding proteins such as TIA1, G3BP1, and PABP, which can block the translation of host and viral proteins, allowing the cell to better adapt to environmental stress. However, whether TIAR affects HBV replication has not been reported.

[0005] The information in the background section is only for the purpose of promoting an understanding of the general background of the application. It should not be taken as an acknowledgement or any form of suggestion that it forms the prior art of this application. SUMMARY

[0006] The present application is based on the finding that TIAR can specifically bind to pgRNA of HBV to promote Pol translation and subsequent pgRNA packaging and viral replication, and that targeting knockdown of TIAR can inhibit HBV replication. The present application is completed based on at least the above finding, and specifically, the present application comprises the following.

[0007] In a first aspect, the present application provides a use of a TIAR inhibitor in the manufacture of a medicament for treating hepatitis B virus infection.

[0008] In some embodiments, according to the use of the first aspect of the present application, wherein the TIAR inhibitor is an inhibitor of TIAR activity or an inhibitor of TIAR expression, or an oligonucleotide that inhibits the binding of TIAR to a target gene;

[0009] Preferably, the oligonucleotide comprises a sequence of the epsilon stem-loop structure of the pgRNA of hepatitis B virus.

[0010] Preferably, the oligonucleotide comprises at least one sequence selected from the group consisting of CUG(C / U)UC sequence, CUGUUC sequence and CUGUGC sequence.

[0011] In a second aspect, the present application provides a method for in vitro regulating the activity of hepatitis B virus, comprising the step of regulating the activity of hepatitis B virus by changing the amount or binding activity of TIAR. In the present application, in vitro includes in ex vivo tissues or cells, and also includes in simulated tissues or cells and in cell-free reaction systems, etc.

[0012] In some embodiments, according to the method of the second aspect of the present application, wherein the viral activity is replication activity, and the replication of hepatitis B virus is up-regulated, enhanced or increased by increasing the amount or binding activity of TIAR, preferably the replication of hepatitis B virus in tissues or cells is increased by overexpressing TIAR in the tissues or cells; or

[0013] The replication of hepatitis B virus is down-regulated, slowed down, reduced or blocked by reducing the amount or binding activity of TIAR, preferably the replication of hepatitis B virus in tissues or cells is down-regulated or blocked by inhibiting or blocking the expression or binding activity of TIAR in the tissues or cells.

[0014] In certain embodiments, the method according to the second aspect of the present application, wherein the viral activity is the expression activity of Pol protein, and the expression of hepatitis B virus Pol protein is up-regulated, enhanced or increased by increasing the amount of TIAR or enhancing its binding activity, preferably the expression of hepatitis B virus Pol protein in the tissue or cell is increased by over-expressing TIAR in the tissue or cell; or

[0015] the expression of hepatitis B virus Pol protein is down-regulated, slowed down, decreased or blocked by decreasing the amount of TIAR or its binding activity, preferably the expression of hepatitis B virus Pol protein in the tissue or cell is down-regulated or blocked by inhibiting or blocking the expression of TIAR or inhibiting its binding activity in the tissue or cell.

[0016] In certain embodiments, the method according to the second aspect of the present application, wherein the viral activity is the translation activity of pgRNA, which in turn alters the expression balance of nucleocapsid protein and Pol protein.

[0017] In certain embodiments, the method according to the second aspect of the present application, wherein the viral activity is the level of its pgRNA packaged into nucleocapsid. Preferably, the level or amount of hepatitis B virus pgRNA packaged into nucleocapsid in the tissue or cell is increased by over-expressing TIAR in the tissue or cell in vitro.

[0018] The third aspect of the present application provides a method for screening a substance useful for treating hepatitis B virus infection, wherein comprising the steps of:

[0019] a step of measuring a first measurement value of hepatitis B virus activity in a cell model;

[0020] a step of administering the substance to be tested to the cell model;

[0021] a step of measuring a second measurement value of hepatitis B virus activity from the cell model after administering the substance to be tested;

[0022] a step of comparing the first measurement value and the second measurement value;

[0023] wherein the cell model over-expresses TIAR.

[0024] In certain embodiments, the method for screening a substance useful for treating hepatitis B virus infection according to the third aspect of the present application, wherein:

[0025] When the viral activity is the replication activity and / or the Pol protein expression activity, and the second measurement value is less than the first measurement value, or when the viral activity is the nucleocapsid protein expression activity, and the second measurement value is greater than the first measurement value, the substance to be tested is screened as a substance useful for treating hepatitis B virus infection. Preferably, the substance to be tested comprises a compound or a composition thereof, a traditional Chinese medicine extract, or a biological macromolecule.

[0026] The present application demonstrates that the host factor TIAR enhances the replication of HBV, and this enhancement is achieved by TIAR binding to pgRNA to regulate its translation, i.e. inhibiting the translation of nucleocapsid protein but promoting the translation of Pol. It is also found that TIAR can also be packaged into the nucleocapsid of HBV by binding to pgRNA. The present application can inhibit the replication of HBV by reducing the expression of TIAR or inhibiting the binding of TIAR to pgRNA, thereby providing a new scheme for treating HBV infection based on TIAR as a target. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Results of exogenous overexpression of TIAR in Huh-7 cells. The results show that overexpression of TIAR promotes the replication of HBV in the HBV in vitro replication cell model and the HBV in vitro infected cell model.

[0028] Figure 2 Results of knockdown of endogenous TIAR in Huh-7 cells. The results show that knockdown of endogenous TIAR inhibits the replication of HBV in the HBV in vitro replication cell model and the HBV in vitro infected cell model.

[0029] Figure 3 Results of RT-qPCR detection of precipitated RNA after co-incubation of specific antibodies of TIAR with lysates of HepAD38 cells stably producing toxins or Huh7 cells transfected with prcccDNA / pCMV-Cre recombinant plasmid system.

[0030] Figure 4 Results of transcriptome and protein translation analysis of HepAD38 cells transfected with siTIAR or control siNC.

[0031] Figure 5 Analysis of the binding site of TIAR and pgRNA. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0033] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, solvent amounts, and the like, it is to be understood that each intervening value, to the upper and lower limits of the ranges is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0034] Unless defined otherwise, 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 application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference for the disclosure and

[0035] As used herein, the term "TIAR" refers to T cell intracellular antigen 1 related protein, which is a key protein constituting the classical stress granule of eukaryotic cells, and the present application finds that it is an RNA binding protein and has specificity for binding to specific RNA sequences.

[0036] As used herein, the term "TIAR inhibitor" refers to a molecule capable of binding to TIAR or its mRNA and affecting its activity (i.e., a TIAR binding molecule), or a molecule capable of inhibiting the expression of TIAR. Typically, when a TIAR binding molecule binds to TIAR or its mRNA, it will typically reduce, attenuate, or completely block the binding activity or expression level of TIAR. Examples of TIAR inhibitors include, but are not limited to, biological macromolecules such as antibodies or active fragments thereof, nucleic acids, oligonucleotides, and small molecule compounds.

[0037] As used herein, the term "TIAR binding activity" refers to the ability or property of TIAR to bind to a target region of HBV genes, such as pgRNA. Preferred target regions include the F1 region of pgRNA, and more preferably the epsilon stem loop structure thereof. Exemplary target region sequences include, but are not limited to, at least one selected from the group consisting of CUG(C / U)UC sequences, CUGUUC sequences, and CUGUGC sequences.

[0038] As used herein, the term "antibody" includes polyclonal, monoclonal, chimeric, nanobody, humanized, or fully human antibodies, as well as antibody modifications or active fragments of such antibodies. The antibodies of the present application are preferably single chain antibodies.

[0039] The term "antibody modifier" herein includes chemical modifiers and conjugates of antibodies and other materials. Examples of chemical modifiers include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, cross-linking cyclization, disulfide bond formation, demethylation, covalent cross-linking, cysteinylation, pyroglutamylation, formylation, gamma-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolysis, and phosphorylation of antibodies, among others. Examples of conjugates include, but are not limited to, conjugates with nanopolymeric materials, magnetic beads, and the like.

[0040] The term "antibody active fragment" herein, which is also sometimes referred to herein as "antigen binding fragment", refers to, but is not limited to, scaffold structures containing one or more complementarity determining regions (CDRs) of an antibody, scaffold structures containing one or more variable domains (of either heavy or light chain), antibody fragments and variants having TIAR specific binding ability. Examples of antibody active fragments or antigen binding fragments include, but are not limited to, Fab fragments, Fab', F(ab')2 fragments, single chain variable fragments scFv, scFv Fc fragments, or single chain antibodies ScAb.

[0041] The antibodies of the present application can be prepared by known methods. In exemplary embodiments, the method of preparing the antibodies includes the step of immunizing an animal with an immunizing antigen, wherein the immunizing antigen is TIAR or a derivative, fragment thereof. Examples of TIAR derivatives include, but are not limited to, conjugates of TIAR or fragments thereof with carrier proteins. Examples of carrier proteins of the present application include, but are not limited to, serum albumin (BSA), chicken ovalbumin (OVA), and keyhole limpet hemocyanin (KLH).

[0042] The term "nucleic acid" herein includes ribonucleic acids and / or deoxyribonucleic acids. The term "oligonucleotide" herein refers to a plurality of linked nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose)) linked to organic bases, which are either pyrimidines (e.g., cytosine (C), thymine (T), or uracil (U)) or purines (e.g., adenine (A) or guanine (G)). Oligonucleotides include DNA (e.g., D- and L-DNA) and RNA, as well as various modifications thereof. Modifications include base modifications, sugar modifications, and backbone modifications. It is to be understood that the oligonucleotides described in the present application can be homogeneous or heterogeneous in nature. For example, they can be entirely DNA or RNA, or a mixture of the two.

[0043] The oligonucleotides of the present application can include modifications to make the oligonucleotides more stable and / or less susceptible to degradation under certain conditions. For example, in some cases, the oligonucleotides are nuclease resistant. In some cases, the oligonucleotides have phosphorothioate backbone modifications that make the oligonucleotides less susceptible to nucleases and more stable (as compared to natural phosphodiester backbone nucleic acids). In some cases, other linkages that can provide higher stability to the oligonucleotides include, but are not limited to, phosphorodithioate linkages, methylphosphonate linkages, methylphosphorothioate linkages, boranophosphonate linkages, peptide linkages, alkyl linkages, phosphodiester linkages, and the like. Thus, in some cases, the oligonucleotides have non-naturally occurring backbones.

[0044] The oligonucleotides of the present application can include modifications in their bases. Modified bases include modified cytosines (e.g., 5-substituted cytosines (e.g., 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and 5-alkynyl-cytosine unsubstituted or substituted), 6-substituted cytosines, N4-substituted cytosines (e.g., N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudoisocytosine, cytosine analogs with fused ring systems (e.g., N,N'-propylene cytosine or phenoxazine), and uracil and its derivatives (e.g., 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil), modified guanines, e.g., 7 deaza guanine, 7 deaza 7 substituted guanine (e.g., 7 deaza 7 (C2 C6) alkynyl guanine), 7 deaza 8 substituted guanine, inosine, N2 substituted guanine (e.g., N2-methyl-guanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6 diaminopurine, 2 aminopurine, purine, indole, adenine, substituted adenines (e.g., N6-methyl-adenine, 8-oxo-adenine), 8 substituted guanines (e.g., 8 hydroxy guanine and 8 bromo guanine), and 6 thioguanine.

[0045] Examples

[0046] I. Experimental Methods

[0047] 1.1 Fluorescent Quantitative PCR

[0048] Dilute 20 μΐ of cDNA product to 100 μΐ with double distilled water, mix well. Take 2 μΐ as template for real-time fluorescent quantitative PCR, use Roche Light Cycle 480II PCR instrument for detection. Reaction conditions: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, 72℃ for 30 s, 40 cycles. Analyze the specificity of primers by melting curve. According to the threshold cycle number Ct value of each reaction well, use 2 -△△Ct Method, β-actin as internal reference, calculate the relative expression of the gene.

[0049] 1.2 Western blotting experiment

[0050] Cells were lysed in pre-chilled RIPA buffer (Invitrogen, California, USA) containing enzyme inhibitors (Roche, Basel, Switzerl). The cell lysate was then centrifuged at 12000 rpm for 10 min at 4℃, the supernatant was collected and the protein concentration was determined by BCA protein assay reagent (Pierce, Illinois, USA). The protein lysate was separated on NuPAGE Bis-Tris gel (Invitrogen, California, USA) and transferred to PVDF membrane (Millipore, Massachusetts, USA). Hybridization was performed with antibodies. The protein-antibody complex was observed using Odyssey Imager (LI-COR Biosciences, North Carolina, USA).

[0051] 1.3 Transfection of siRNA

[0052] Cells in logarithmic growth phase were trypsinized, counted and inoculated into 6-well culture plates at a density of 3 x 10 5 Two RNase-free EP tubes were taken, 600 μΐ of Opti-MEM solution was added to each, 180 pmols of siRNA was added to one, and 15 μΐ of Lipofectamine RNAi MAX was added to the other. After mixing, they were placed at room temperature for 5 min. The solutions in the two tubes were mixed, mixed gently, and placed at room temperature for 20 min. The mixed solution was added to the 6-well culture plate, 200 μΐ per well. Incubate in a 5% CO2, 37℃ incubator.

[0053] 1.4 Ribo-seq

[0054] Knockdown and overexpression of TIAR in HepAD38 in vitro replication cell model and HepG2-NTCP in vitro infection cell model, 48h after the addition of cycloheximide fixation and harvest cells, entrust Guangzhou Kidio Company to carry out ribosome spectrum analysis detection.

[0055] 1.5 Northern blot hybridization

[0056] Harvest the cell precipitate and HBV replication mouse liver tissue of different treatment groups and control groups of HBV in vitro replication model and in vitro infection model, and extract the tissue or cell RNA by using TRIZOL reagent. 5 μg of total RNA is denatured, prepared into an agarose gel, and electrophoresed at 70 V constant voltage for 5-7 h. After transfer to nylon membrane, UV cross-linking fixation, pre-hybridization, probe hybridization, blocking and antibody incubation are carried out in sequence, and comparison analysis is carried out by ECL development.

[0057] 1.6 Cell immunofluorescence experiment

[0058] The cells are inoculated in a 24-well plate, and are sequentially fixed with 4% paraformaldehyde, permeated with 0.2% Triton X-100, and blocked with goat serum at room temperature. Finally, the primary antibody of the target protein, fluorescent secondary antibody are incubated, DAPI solution is added to stain the nucleus, and observation and photography are carried out under a confocal microscope.

[0059] II. Experimental results

[0060] 2.1 Expression of exogenous TIAR promotes HBV replication

[0061] In order to study the role of TIAR in HBV replication, prcccDNA / pCMV-Cre plasmid was transiently transfected in Huh-7 cells, and TIAR was overexpressed. The results show that overexpression of TIAR significantly increases the HBV DNA level in the culture supernatant and the capsid, but reduces the level of nuclear capsid protein ( Figure 1 A-C). At the same time, overexpression of TIAR does not change the level of HBV RNA transcript ( Figure 1 D), the level of HBsAg and HBeAg in the culture supernatant ( Figure 1 E-F) and the level of L-HBs in the cells ( Figure 1 C). In addition, similar results are observed in HepAD38 cells ( Figure 1 H-L) and HBV in vitro infection cell model ( Figure 1 M-R). In summary, overexpression of TIAR is confirmed to promote HBV replication in HBV in vitro replication cell model and HBV in vitro infection cell model.

[0062] 2.2 Knockdown of TIAR inhibits HBV replication

[0063] To further confirm the inhibitory role of TIAR in HBV replication, prcccDNA / pCMV-Cre plasmid was transiently transfected into Huh-7 cells, and endogenous TIAR was knocked down using siRNA. The results showed that TIAR knockdown significantly reduced HBV DNA levels in the culture supernatant and capsid, but increased nucleocapsid protein levels. Figure 2 AC). Meanwhile, knocking down TIAR did not alter HBV RNA transcript levels ( Figure 2 D) Levels of HBsAg and HBeAg in the culture supernatant ( Figure 2 EF) and intracellular L-HBs levels ( Figure 2 C). Furthermore, in HepAD38 cells ( Figure 2 HL) and HBV in vitro infection cell model ( Figure 2 Similar results were observed in MR. In summary, knockdown of endogenous TIAR was confirmed to inhibit HBV replication in both in vitro HBV replication cell models and in vitro HBV infection cell models.

[0064] 2.3 TIAR can bind to pgRNA and be encapsulated in the nucleocapsid.

[0065] IgG is the most abundant immunoglobulin in the human body and is usually used as a control antibody in immunoprecipitation experiments. To confirm the interaction between TIAR and pgRNA, TIAR-specific antibodies and homologous IgG were co-incubated with lysates of stable toxin-producing HepAD38 cells or lysates of Huh7 cells transfected with the prcccDNA / pCMV-Cre recombinant plasmid system. The precipitated RNA was then detected by RT-qPCR. The results showed that the pgRNA levels precipitated by TIAR antibody were significantly higher than those precipitated by control IgG. Figure 3 A). Biotin-labeled pgRNA was co-incubated with HepG2 cells and pulled using streptavidin magnetic beads. Results showed that the biotin-labeled pgRNA could bind to TIAR ( ) in cell lysate in vitro. Figure 3 B) further confirmed the binding of TIAR to pgRNA. To confirm that TIAR is encapsulated in the nucleocapsid, sucrose density gradient centrifugation analysis was performed on the culture supernatant of HepAD38 cells, and the results showed that TIAR and HBV DNA settled in the same layer (B). Figure 3 C). This proves that TIAR is encapsulated in a nucleocapsid.

[0066] 2.4 TIAR promotes pgRNA translation of P protein

[0067] TIAR had no effect on the levels of all HBV transcripts, but its effects on the levels of HBV DNA in culture supernatant and nuclear capsid protein in cells were opposite.Figures 1-2 ). It was reported that increasing the translation of Pol protein could promote HBV replication, thus it was speculated that TIAR might change the balance of expression of nucleocapsid protein and Pol protein by regulating the translation of pgRNA, and then regulate the replication of HBV.

[0068] To verify this hypothesis, HepAD38 cells transfected with siTIAR or control siNC were lysed, and then polysomes were isolated, followed by nuclease treatment, which could digest the RNA not protected by ribosomes, while the RNA fragments undergoing translation were protected by ribosomes and not digested (about 30 bases) Figure 4 A). First, the effect of TIAR on the translation level of HBV RNA was analyzed by the method of ribosome profiling (Ribo-seq). The results showed that there was no significant difference in HBV transcripts between siTIAR and siNC transfected HepAD38 cells Figure 4 B), so the difference in ribosome footprints (RF) between the two groups could reflect the difference in translation. Further, the P-site of each RF was inferred using the RiboWave data analysis tool to accurately locate each ORF. The results showed that compared with the control group of cells, the RF count on the ORF of nucleocapsid protein was higher, while the RF count on the ORF of Pol protein was lower in HepAD38 cells with endogenous TIAR knocked down, especially in the starting region of the ORF of Pol protein Figure 4 C-D), indicating that knocking down endogenous TIAR promoted the translation of nucleocapsid protein but inhibited the translation of Pol protein. In addition, there was no significant difference in the translation of HBsAg between the control and experimental groups of HepAD38 cells, which was consistent with the previous results that knocking down or overexpressing TIAR did not change the level of secreted HBsAg Figures 1-2 ).

[0069] To further verify the effect of TIAR on the translation of pgRNA, siNC or siTIAR was transfected into HepAD38 cells, and protein quantification mass spectrometry based on parallel reaction monitoring (PRM) was performed. Consistent with the results of translation group, the abundance of nucleocapsid protein significantly increased, while the abundance of Pol protein significantly decreased after knocking down endogenous TIAR, and the abundance of HBsAg did not change significantly Figure 4 E). In addition, in order to directly determine the effect of TIAR on the translation of pgRNA, a truncated pgRNA plasmid was constructed, which could encode full-length nucleocapsid protein and truncated Pol protein with 3xflag tag at the C-terminal Figure 4F). The results showed that knockdown of endogenous TIAR significantly reduced the expression of PolAC-flag and increased the expression of core protein in Huh-7 cells transfected with plasmids of truncated pgRNA, and overexpression of exogenous TIAR also reached the same conclusion Figure 4 G).

[0070] In summary, TIAR changes the balance of core protein and Pol protein by changing the translation of pgRNA, promotes the expression of Pol, and then promotes the packaging of pgRNA, the replication of viral DNA and the production of progeny virus, thereby promoting HBV replication.

[0071] 2.5 TIAR promotes the translation of Pol protein by binding to the stem-loop structure of pgRNA

[0072] To explore the binding site of TIAR and pgRNA, the HBV genome sequence was divided into five segments Figure 5 A), and the results of RNA pulldown experiment showed that TIAR had the strongest binding ability with the transcript of F1 region Figure 5 B). In order to further explore the exact binding site of TIAR in pgRNA, cross-linking immunoprecipitation experiment (CLIP-qPCR) was performed Figure 5 C). Western blot experiment confirmed that the specific antibody precipitated TIAR-flag protein Figure 5 D). The results proved that TIAR could bind to the F1 region of HBV RNA Figure 5 F and Figure 5 G). In addition, another primer (primer ε) was designed between the target regions of primers 1 and 2 to further clarify the binding sequence. The target sequence of primer ε precipitated by TIAR could not be detected obviously Figure 5 F), which indicated that TIAR combined with the ε stem loop of pgRNA. The bulge loop (CUGUUC) and loop loop (CUGUGC) on the ε stem loop were similar to the previously reported TIAR binding sequence CUG(C / U)UC sequence Figure 5 G). After mutating ε or bulge and loop in F1 fragment at the same time, it was found that the deletion of ε led to the disappearance of the interaction between TIAR and F1 region RNA; while only deleting bulge or only deleting loop, TIAR could still bind to F1 region RNA, but the binding ability was significantly reduced, especially the loop deletion Figure 5 H). Therefore, it was judged that the binding of TIAR to pgRNA depended on the bulge and loop loops of ε stem loop. Further, mutating the bulge and loop loops of ε stem loop in truncated pgRNA plasmid, it was found that changing the expression of TIAR no longer affected the expression of core protein and Pol ​I), suggesting that TIAR promotes pgRNA translation of the Pol protein by binding to the stem-loop structure of pgRNA.

[0073] While the application has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes can be made and equivalents can be substituted for elements thereof without departing from the scope or spirit of the application. In addition, many modifications can be made to the example embodiments of the disclosure in light of the teachings below. The scope of the claims should be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. A method for screening useful substances, characterized by, The method comprises the following steps: a step of measuring a first measurement value of hepatitis B virus activity in a cell model; a step of applying a test substance to the cell model; a step of measuring a second measurement value of hepatitis B virus activity in the cell model after the test substance is applied; a step of comparing the first measurement value and the second measurement value; wherein the cell model is a cell model overexpressing an exogenous TIAR; the hepatitis B virus activity is the expression activity of nucleocapsid protein, and when the second measurement value of the expression activity of nucleocapsid protein of hepatitis B virus is greater than the first measurement value, the test substance is screened as a substance useful for treating hepatitis B virus infection.

2. The method for screening useful substances according to claim 1, characterized by, The test substance is a compound or a composition thereof, a traditional Chinese medicine extract, or a biological macromolecule.

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