Specific host factors of hepatitis B virus infection and their applications

Abstract

Involving specific host factors of hepatitis B virus infection, it was found that the specific host factor CREBH can significantly enhance hepatitis B virus infection. On the one hand, it can promote the invasion of hepatitis B virus, and at the same time, it also has a certain enhancement on the transcription of hepatitis B virus. By discovering and identifying specific host factors, the scientific community has expanded its understanding of the hepatitis B virus invasion process and found a new target for inhibiting hepatitis B virus infection.

Description

Technical Field

[0001] The present invention relates to the fields of disease mechanism research and drug development, and in particular to specific host factors of hepatitis B virus infection and applications thereof. Background Art

[0002] Hepatitis B virus (HBV) is one of the most widespread viruses infecting people worldwide. According to data from the World Health Organization in 2018, 257 million people remain infected worldwide, and in 2015 alone, 880,000 people died from HBV-related diseases. However, there is currently no targeted drug that can cure HBV infection. Commonly used drugs for treating HBV include interferon-α (IFN-α) and nucleotide analogs such as entecavir. The HBV genome consists of approximately 3.2 kb of open double-stranded DNA (dsDNA), containing a complete negative strand and a partial positive strand, known as relaxed closed circular DNA (rcDNA). After infecting a cell, the positive strand is first repaired to form covalently closed circular DNA (cccDNA). cccDNA can be stably present in the host cell nucleus and serves as a template for transcription of all viral genes. The difficulty of eliminating cccDNA has become a core issue in curing HBV infection. The HBV genome itself is extremely simple, and viral replication and life processes are all completed under the coordinated action of host factors.

[0003] In 1993, Gripon et al. found that the addition of polyethylene glycol (PEG) significantly increased the efficiency of hepatitis B virus infection in an in vitro PHH infection system. However, adding PEG after the virus bound to cells did not promote infection. Therefore, it is speculated that PEG promotes the binding of the virus to cell membrane receptors rather than enhancing fusion of the virus with the cell membrane. Furthermore, it is known that the viral titer required for in vitro infection is much higher than the minimum viral dose required for infection in gorilla infection models or under physiological conditions. HBV requires specific host factors to promote viral infection. Summary of the Invention

[0004] The first object of the present invention is to provide a specific host factor for hepatitis B virus infection, which has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence that is at least 90% identical to the amino acid sequence as shown in SEQ ID NO: 1 and has activity as a host factor for hepatitis B virus infection.

[0005] Existing in vitro hepatitis B virus infection methods, such as those for HepG2-NTCP cell lines or primary human liver cells (PHH), require the simultaneous addition of 4%-5% PEG to enhance infection. The addition of PEG does not induce virus-cell fusion, but rather increases contact between the virus and HSPG or NTCP. Even so, a high titer of virus must be added simultaneously to achieve efficient infection. In contrast to in vitro infection, in vivo infection does not require these conditions. Based on the significant differences in infection conditions, it is not difficult to infer that in in vitro infection systems, even after binding to NTCP, the virus still lacks host factors that facilitate viral invasion. To discover and identify unknown host factors that restrict viral invasion, the present invention first constructed a cDNA expression library based on 74 liver-specifically expressed genes defined by the HPA database. The present invention selected infection at a 1% PEG concentration and screened the cDNA library to identify the CREBH gene. The protein expressed by the CREBH gene has the amino acid sequence shown in SEQ ID NO:1.

[0006] The CREBH (cAMP-responsive element-binding protein, hepatocyte specific) gene was first cloned and identified in 2001. Due to its characteristic bZIP domain with high homology, it was classified into the CREB / ATF gene family. Compared with the ubiquitous expression of other genes in the same family, CREBH has liver-specific expression characteristics, hence its name. Subsequent experiments found that CREBH is also expressed in small intestinal tissue. CREBH has a similar structure to the gene ATF6 in the same family. Both genes encode a transmembrane region within the gene and are type II transmembrane proteins. After expression, the full-length gene is localized to the endoplasmic reticulum or Golgi membrane, while the protein lacking the transmembrane region is localized to the cell nucleus. In 2006, Kezhong Zhang et al. demonstrated that CREBH can be transferred from the endoplasmic reticulum to the Golgi apparatus via the RIP (regulated intramembrane proteolysis) pathway, where it is cleaved by S1P (Site 1 protease) and S2P (Site 2 protease), releasing the amino-terminal domain of the protein to enter the nucleus and activate gene expression. Similar genes include SREBPs (sterol regulatory-element binding proteins), key regulators of cholesterol and lipid metabolism.

[0007] As a preferred embodiment of the present invention, the specific host factor is a specific host factor for hepatitis B virus invasion of cells, or a specific host factor for hepatitis B virus transcription, or a specific host factor for hepatitis B virus invasion of cells and viral transcription. Specifically, in experiments examining the role of CREBH following hepatitis B virus infection, the present invention first confirmed the necessity of the transcriptional activation domain by cloning truncated CREBH. Secondly, combined with the results of a hepatitis B virus Northern blot in a CREBH-overexpressing cell line, it was confirmed that CREBH can alter the transcriptional state of hepatitis B virus. This effect was more pronounced under 5% PEG conditions, likely due to the increased formation of cccDNA during infection, meaning that CREBH has more transcriptional templates to influence. However, compared to experiments in which CREBH was transduced after infection, the promoting effect of CREBH transduced before infection was more pronounced under 1% PEG infection conditions, indicating that CREBH promotes viral invasion. This result demonstrates the dual functionality of CREBH, namely, promoting viral invasion and enhancing hepatitis B virus transcription. In addition, judging from the HBcAg results of hepatitis B virus infection, CREBH transduction can increase the number of stained cells, and CREBH can significantly increase the infection of hepatitis B virus at low virus titers, both of which support that CREBH promotes viral invasion.

[0008] The present invention also protects the activating factor of the specific host factor. As a preferred embodiment of the present invention, the activating factor is insulin. The CREBH gene is a transmembrane protein and also has the ability to activate transcription. The gene structure of ATF6, the gene with the highest homology to CREBH, is roughly the same and needs to be cleaved to release the amino-terminal domain to function. However, compared with ATF6, the carbon-terminus of CREBH is much shorter, and the function of the domain is unknown. Previous studies have reported that the carbon-terminal domain of the ATF6 gene is necessary for responding to upstream ER stress signals, suggesting that CREBH and ATF6 may be induced and activated in response to different signal sources. However, in the CREBH screening experiment and subsequent verification experiments, apart from using PMM culture medium, no treatment that can activate CREBH was specifically added. The staining results of CREBH and its truncated form confirmed the separation of its amino-terminal and carbon-terminal staining signals, indicating that in the experiment of transducing CREBH to infect hepatitis B virus, CREBH had been cleaved and activated. Although there are reports that CREBH can be partially spontaneously activated when driven by a strong promoter such as the CMV promoter, most experiments on CREBH have also been conducted under exogenous strong promoters and no significant self-activation has been observed. Therefore, the activation after CREBH transduction in the present invention is more likely to come from the stimulation of PMM culture medium. The present invention verifies that DMSO and insulin in PMM may be the signal sources for CREBH activation.

[0009] The present invention also protects the truncated form of the specific host factor, preferably the amino-terminal domain of the specific host factor. As a preferred embodiment of the present invention, the amino-terminal domain is residues 1-122 of the CREBH protein (such as the amino acid sequence shown in SEQ ID NO: 2), or residues 122-318 (such as the amino acid sequence shown in SEQ ID NO: 3), or residues 1-122 and residues 122-318. As a preferred embodiment of the present invention, the amino-terminal domain is residues 211-318 of the CREBH protein (such as the amino acid sequence shown in SEQ ID NO: 4). Specifically, in order to analyze the promoting effect of each domain of CREBH on hepatitis B virus infection, the present invention truncated CREBH. The experiment of infection with the truncated mutant clearly shows that the transcriptional activation and DNA binding domains of CREBH are necessary. But unexpectedly, although the CREBH-122 clone has removed part of the transcriptional activation function in structure and function, it has the strongest promoting effect on hepatitis B virus infection. By analyzing the results of mRNA sequencing, it was found that there were 109 genes that were upregulated more than 2 times after transduction of CREBH, and 104 genes that were upregulated more than 2 times after transduction of CREBH-122, but there were only 17 genes with consistent changes. This may be due to the regional distribution of the CREBH amino-terminal transcriptional activation domain, but it cannot be ruled out that the downstream gene activation caused by CREBH-122 is non-natural activation. About the impact of CREBH truncated clones on infection, the present invention, except that CREBH-122 and CREBH-122-318 clones significantly enhanced infection, also observed that the shorter truncated CREBH-211-318 slightly inhibited hepatitis B virus infection. Because the bZIP domain in CREBH needs to dimerize to become homologous or heterodimers and bind DNA, and the CREBH-211-318 clone only expressed the bZIP domain, it is speculated that this truncated form of CREBH can form a non-functional dimer with wild-type CREBH, with a dominant inhibitory effect. This result is consistent with the finding that knocking down CREBH inhibited infection.

[0010] The expression and cell membrane localization of NTCP are key factors in hepatitis B virus infection. Therefore, the factors affecting infection in the present invention must first determine whether they alter NTCP. CREBH transduction downregulates NTCP transcription levels, but since NTCP expression in HepG2-NTCP cells is driven by the CMV promoter, it is unclear how CREBH affects the CMV promoter. CREBH does not alter the cell membrane localization of HepG2-NTCP-GFP. Myr47 polypeptide also inhibits hepatitis B virus infection in cells transduced with CREBH or in the PWPI empty-load control group, suggesting that CREBH may be a relatively independent infection-restricting factor located downstream of NTCP.

[0011] Based on the above research and findings, the present invention simultaneously protects the nucleotides encoding the specific host factor or the truncated form of the specific host factor, as well as the vector into which the nucleotides are inserted and / or which is capable of exogenously expressing the specific host factor or the truncated form of the specific host factor.

[0012] The present invention further protects cells susceptible to hepatitis B virus infection into which the isolated nucleotide or vector has been exogenously introduced. Preferably, the cells are selected from HepG2 cells, HepG2-NTCP cells, and primary human hepatocytes (PHH). To achieve CREBH activation, the cells are preferably cultured in a medium containing DMSO and / or insulin, preferably PMM medium.

[0013] The present invention further protects the use of the nucleotide or vector in constructing a non-human transgenic animal model with CREBH gene knockout or exogenous expression of CREBH gene, and in constructing a non-human transgenic animal model infected with or susceptible to hepatitis B virus infection.

[0014] The present invention further protects the use of the specific host factor, the truncated form, the activating factor, the nucleotide, the vector, the cell, or a non-human transgenic animal model constructed using the nucleotide or vector in screening drugs for treating and / or preventing hepatitis B virus infection and / or related diseases.

[0015] The present invention further protects a method for screening drugs for treating and / or preventing hepatitis B virus infection or related diseases, which comprises: applying the specific host factor, the truncated form, the activating factor, the nucleotide, the vector, the cell, or a non-human transgenic animal model constructed using the nucleotide or vector.

[0016] The present invention further protects the use of the specific host factor, the truncated form, the activating factor, the nucleotide, a substance that prevents or reduces the protein expression / function of the specific host factor or the truncated form, a substance that prevents or reduces the interaction between the specific host factor or the truncated form and the hepatitis B virus, and / or a substance that prevents or reduces the transcription of the nucleotide in treating and / or preventing hepatitis B virus infection and related diseases in mammals.

[0017] The present invention further protects a drug for treating and / or preventing hepatitis B virus infection and related diseases in mammals, wherein the drug comprises: the specific host factor, the truncated form, the activating factor, the nucleotide, a substance that prevents or reduces the protein expression / function of the specific host factor or the truncated form, a substance that prevents or reduces the interaction between the specific host factor or the truncated form and the hepatitis B virus, and / or a substance that prevents or reduces the transcription of the nucleotide.

[0018] As a preferred embodiment of the present invention, the drug comprises residues 211-318 of the CREBH protein.

[0019] As a preferred embodiment of the present invention, the substance that reduces the transcription of the nucleotide can be dsRNA, siRNA, RNA interference vector or RNA interference lentivirus targeting the above-mentioned isolated nucleotide.

[0020] As a preferred embodiment of the present invention, the isolated siRNA of nucleotide is selected from:

[0021] siCREBH-1:gcugcuggaaagauggcuu as shown in SEQ ID NO:5

[0022] siCREBH-2 as shown in SEQ ID NO: 6: gcuccuggaucuccuguuu

[0023] siCREBH-3:cccucuuggagcaacugaa as shown in SEQ ID NO:7.

[0024] As a preferred embodiment of the present invention, the drug comprises: an insulin inhibitor.

[0025] Furthermore, the present invention demonstrates that CREBH does not promote infection with hepatitis D virus (HDV). This phenomenon can be explained from the following perspectives. First, the present invention found that HDV infection is more dependent on the expression of NTCP on the cell membrane than hepatitis B virus infection, and that HDV is more easily infected at low PEG concentrations. Although HDV and hepatitis B virus share the same viral envelope, HDV can still package the virus in the absence of the L protein, indicating significant differences between the two during the later stages of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the cDNA library screening result in Example 1.

[0027] Figure 2 Schematic diagram of the construction of the CREBH truncated clone in Example 2.

[0028] Figure 3 The effect of the truncated form of CREBH on HBV infection in Example 2; wherein, A, HbeAg when infected with 1% PEG; B, HBsAg when infected with 1% PEG; C, HbeAg when infected with 5% PEG; D, HBsAg when infected with 5% PEG.

[0029] Figure 4 The figures show the infection results of transduced CREBH and its truncated clones at different virus titers in Example 2; A, 1% PEG infection results; B, 5% PEG infection results; C, four experimental groups: 1% PEG CREBH-122, 1% PEG PWPI, 5% PEG, and 5% PEG CREBH-122. The HBeAg value at day 6 at 100% viral infection was calculated as 1, and the HBeAg value of each group at 100% viral infection was divided by the HBeAg value at 100% viral infection to obtain the relative infection rate diagram.

[0030] Figure 5 Schematic diagram of the effects of transduction of CREBH and its truncated form after HBV infection in Example 2; wherein, A, 1% PEG HbeAg; B, 1% PEG HbsAg; C, 5% PEG HbeAg; D, 5% PEG HBsAg.

[0031] Figure 6 Schematic diagram of the effect of knocking down CREBH on infection in Example 4; A, 48 hours after HepG2-NTCP cells were transfected with three CREBH siRNAs, RNA was collected by TRIzol and the relative expression level of CREBH was detected by reverse transcription; B, HBeAg in the supernatant after HBV infection; C, HBsAg in the supernatant after HBV infection. DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0033] Example 1: Construction and screening of a hepatocyte membrane protein gene cDNA library

[0034] 1. Effect of PEG on infection

[0035] HepG2-NTCP (AC12) is an existing cell line that can be efficiently infected by HBV. It is a monoclonal cell line that stably expresses NTCP and was screened based on the HepG2 cancer cell line. However, according to existing research and laboratory data, achieving efficient HBV infection in HepG2-NTCP cells still requires the additional addition of PEG (PEG8000) and a viral titer far higher than that required for in vivo infection. It is generally believed that the role of PEG is to increase the contact between the virus and cell membrane surface receptors. The high viral titers used at the same time suggest that host factors that limit infection or lack infection-dependent host factors may exist in the cells cultured in vitro. Therefore, using low viral titers or reducing PEG concentrations for genetic screening for infection may reveal factors that can enhance the interaction of the virus with cell membrane surface receptors, as well as key host factors that are critical for viral invasion.

[0036] The experiment first tested the infection of HepG2-NTCP cells at different PEG concentrations. The results showed that the infection of HepG2-NTCP cells was dependent on the PEG concentration added during infection. 1% PEG and 2% PEG promoted HBV infection, but the degree of infection was far less than that achieved when 5% PEG was present.

[0037] 2. Construction and screening of hepatocyte membrane protein library

[0038] The present invention queried and utilized the Human Protein Atlas database for definitions and groupings of liver-specifically expressed genes. A total of 426 liver-specifically expressed genes were identified. Because host restriction factors affecting infection are presumed to be more likely to be located on the cell membrane or endomembrane system, 114 membrane protein genes were selected from these 426 genes for cloning using annotated subcellular localization information. These genes were then cloned into the PWPI vector using enzyme digestion and ligation. After sequencing verification, a target library containing 76 genes was successfully constructed.

[0039] The cDNA library was prepared by packaging lentivirus pseudovirus into HepG2-NTCP cells expressing cDNA clones. 24 hours later, HBV of the same viral titer was infected under 1% PEG conditions. The culture supernatant was collected every two days and tested using HBeAg and HBsAg kits. The results are shown in Figure 2. Figure 1As shown, clone 15 (CREBH) significantly enhanced HBV infection, and the HBeAg level was comparable to that in 5% PEG. HBcA staining results showed that CREBH significantly increased the number of HBcAg-positive cells, suggesting that CREBH may increase infection efficiency.

[0040] Example 2: CREBH and Hepatitis B Virus Infection

[0041] 1. Expression pattern of CREBH in tissues

[0042] Clone 15 encodes the CREBH (Cyclic AMP Response Element-binding Protein H, CREB3L3) gene. According to information on the CREBH gene in the GTEx database, CREBH is specifically expressed in the liver and small intestine, but its expression level in the liver is much higher than that in the small intestine.

[0043] The experiment also examined CREBH expression in HepG2-NTCP, mPHH (humanized mouse-derived primary hepatocyte) cells, and patient HCC and adjacent adjacent tissues. Expression levels in HepG2-NTCP and mPHH cells were similar, though not high. Expression levels in HCC and adjacent adjacent tissues were significantly higher than in the cell lines, with a slight increase in HCC relative to adjacent adjacent tissues. This suggests that CREBH expression is suppressed in cell lines cultured in vitro, potentially contributing to the lower infection efficiency in vitro compared to in vivo.

[0044] 2. Functional analysis of truncated CREBH

[0045] Human CREBH consists of 461 amino acids and is a type II transmembrane protein. The amino-terminus of CREBH contains a bZIP domain that binds to DNA and a transmembrane region that anchors the protein to the endoplasmic reticulum membrane. Upon stimulation, CREBH is transported to the Golgi membrane, where it is cleaved by Sire 1 and Sire 2 proteases, releasing the amino-terminus with transcriptional activation function and entering the nucleus. The function of the remaining carbon-terminus is unknown. Among its regulatory functions, CREBH has been reported to be involved in lipid and cholesterol metabolism. Due to the inconsistent subcellular localization of its two terminal domains, further investigation is needed to determine which domain is responsible for enhancing hepatitis B virus infection.

[0046] 3. CREBH secondary structure prediction results

[0047] First, the secondary structure of CREBH was predicted using the CFSSP database (Chou and Fasman Secondary Structure Prediction server). Based on the prediction results, three breakpoints were selected in the middle of the CREBH protein, and a total of six truncated clones were constructed, such as Figure 2 shown.

[0048] Secondary structure prediction revealed a low-scoring segment between amino acids 1-122, suggesting a possible intrinsic disorder region. Such regions are often closely associated with transcriptional activation in transcription factors. To maintain the integrity of the bZIP domain, a second breakpoint was selected at amino acid position 211, and a third breakpoint was selected near the transmembrane region at amino acid position 318. Six truncated CREBH clones were constructed. All clones were tagged with an HA tag at the amino terminus and a c-myc tag at the carbon terminus.

[0049] 4. Cellular localization of truncated CREBH

[0050] To investigate the localization of CREBH in the HepG2 cell line, the amino terminus of the CREBH and its truncated forms constructed above were linked to an HA tag, and the carbon terminus was linked to a c-myc tag. HepG2-NTCP cells were transduced with lentivirus, and the HA tag and c-myc tag were stained, respectively, to observe the localization of different truncated forms of CREBH in the cells.

[0051] The staining results show that in the full-length CREBH clone, the staining of the amino terminus and the carbon terminus is slightly separated. A small amount of the amino terminus of CREBH is located in the nucleus, while most of it is retained in the cytoplasm and overlaps with the carbon terminus staining. In the truncated CREBH-122 clone, the separation between the amino terminus localized in the cell nucleus and the carbon terminus localized in the cytoplasm is more obvious. This indicates that compared with the full-length CREBH (as shown in SEQ ID NO: 1), the amino-terminally truncated CREBH-122 (as shown in SEQ ID NO: 2) can be cleaved more efficiently, or the amino-terminal domain of CREBH-122 has better stability in the cell nucleus. CREBH-C (shown in SEQ ID NO:8), CREBH-122-318 (shown in SEQ ID NO:3), and CREBH-211-318 (shown in SEQ ID NO:4) showed similar localization, with the majority located in the nucleus. However, the shorter forms CREBH-307 (shown in SEQ ID NO:9) and CREBH-211-C (shown in SEQ ID NO:10) lost nuclear localization, suggesting that the nuclear localization signal of CREBH may be located between amino acids 211-318.

[0052] To test whether the truncated forms still possess transcriptional activation, the authors used APOA4 gene activation, as reported in the literature, as an indicator of CREBH's transcriptional activation capacity. After transduction of the truncated forms into HepG2-NTCP cells, relative APOA4 expression was measured, revealing that full-length CREBH exhibited the strongest activation of the APOA4 gene. CREBH-C also demonstrated efficient activation, but at approximately one-third the efficiency of full-length CREBH. Although its amino terminus is identical to full-length CREBH, it lacks the transmembrane domain at the carbon terminus, likely due to its impact on protein stability, thereby indirectly reducing APOA4 activation. Furthermore, CREBH-122 and CREBH-122-318 also demonstrated some activation of the APOA4 gene, but their activation abilities were significantly different from those of full-length CREBH and CREBH-C, indicating that the domain responsible for CREBH's transcriptional activation lies between amino acids 1-122.

[0053] 5. Role of truncated CREBH in hepatitis B virus infection

[0054] HepG2-NTCP cells were transduced with lentivirus containing CREBH and its truncated form. After 24 hours, the cells were re-plated on culture plates and the culture medium was changed to PMM. After 24 hours, HBV was infected with 1% PEG and 5% PEG respectively. The supernatant was collected every 2 days and tested with ELISA kit. The infection results were shown in the figure. Figure 3The results show that full-length CREBH, CREBH-122, CREBH-C, CREBH-122-318, and CREBH-211-C all improved infection efficiency at both 1% and 5% PEG infection concentrations compared to the PWPI-B empty control. CREBH-122 showed a significantly higher enhancement effect than the other experimental groups.

[0055] The experiment also included staining for HBcAg, and the results were consistent with those from the ELISA. In addition to the CREBH, CREBH-122, and CREBH-122-318 truncated clones' promotion of HBV infection, a certain degree of inhibition of infection by CREBH-307 and CREBH-211-318 was observed, consistent with the ELISA results. This suggests that these two truncated clones may exert a dominant inhibitory effect.

[0056] In summary, CREBH's enhancing effect on HBV infection depends on its own transcriptional activation function, and CREBH-122 can significantly enhance HBV antigen and the number of HBcAg-positive cells. Therefore, it was used to replace full-length CREBH in subsequent experiments to study the specific mechanism of action.

[0057] 6. Promoting effect of CREBH-122 on infection at different virus titers

[0058] In addition to being affected by PEG, the HBV in vitro infection process also depends on the virus titer. Therefore, the experiment used the most potent promoters, CREBH-122 and CREBH-122-318, and a slightly inhibitory truncated clone of CREBH-211-318, as controls against the PWPI empty vector to test the differences in infection efficiency at different virus titers. If infection occurs under completely ideal conditions, the viral infection index will exhibit a single-factor linear relationship with the amount of virus input. If one or more other restrictive factors are present, the infection curve will deviate from the linear correlation, and infection will decrease significantly as the viral titer decreases.

[0059] Specifically, HepG2-NTCP cells were transduced with lentivirus containing CREBH and its truncated form. After 24 hours, the cells were re-plated onto culture plates and the culture medium was changed to PMM for 24 hours. Based on the normal infection of 160ul virus / 200ul system, the virus titer was calculated as 100%. The virus titers of 50%, 25%, and 12.5% were added to the infection system, and the insufficient volume was made up with PMM. The diluted HBV was infected under 1% PEG and 5% PEG conditions, respectively. The ELISA results of the infection showed that CREBH and its truncated form had the same effect on infection at each virus titer.

[0060] Depend on Figure 4 The results of A show that under the infection condition of 1% PEG, the infection of various truncated types or the control group decreased significantly as the virus titer decreased. Figure 4 The results of B show that under 5% PEG infection conditions, the trend of infection decreasing with decreasing virus titer in cells transduced with CREBH-122 and CREBH-122-318 was alleviated.

[0061] In order to show the results more clearly, the experiment selected the HBeAg of the CREBH-122 and PWPI empty vector groups at 6 days of infection for plotting. The HBeAg of each group at 100% virus infection was calculated as 100%, and the infection value at other titers was divided by it to obtain the percentage of relative infection value. A line graph was drawn with the infection percentage as the vertical axis and the virus titer as the horizontal axis. The results are shown in the figure below. Figure 4 As shown in C. The graph shows that the curves obtained with 1% PEG CREBH-122, 1% PEG PWPI, and 5% PEG infection are similar. However, transduction of CREBH-122 with 5% PEG significantly improved the curves to be closer to linear. Calculating the respective Pearson r values, the correlation with 5% PEG CREBH-122 was also higher (1% PEG CREBH-122 = 0.986, 1% PEG PWPI = 0.968, 5% PEG = 0.986, 5% PEG CREBH-122 = 0.994). This suggests that CREBH-122 transduction overcomes a key limiting factor in HBV infection of HepG2-NTCP.

[0062] 7. The role of CREBH in hepatitis B virus infection

[0063] Since the increased secretion of HBeAg and HBsAg after infection may be due to the enhanced transcription of HBV cccDNA or the enhanced protein secretion pathway, it does not directly reflect the increase in viral invasion. In addition, CREBH itself has a transcriptional activation effect. Therefore, it is necessary to detect the specific steps in which CREBH promotes HBV infection.

[0064] Depend on Figure 5The results show that, first, the experiment transduced CREBH and its truncated clones into the HepG2-NTCP cell line that had been infected with HBV. The effects of CREBH and its various truncated forms on HBeAg and HBsAg were consistent with those before infection, indicating that CREBH may indeed act directly on HBV cccDNA itself. Secondly, HepG2-NTCP cells were infected with HBV for 24 hours and then transduced with letivirus. The supernatant was collected every two days to detect HBeAg and HBsAg. Compared with 1% PEG and 5% PEG infection, the promoting effect of CREBH and its truncated forms under 5% PEG infection conditions was stronger. This may be because more cccDNA was formed in the cells as a template for transcription during 5% PEG infection, which indirectly showed that CREBH had a stronger effect. This further proves that CREBH can enhance the transcription of HBV.

[0065] Example 3: CREBH affects hepatitis B virus infection without changing NTCP

[0066] NTCP is a key receptor in HBV infection, directly binding to the PreS1 region of the virus. Many factors can influence HBV infection, potentially indirectly by affecting NTCP expression or cellular localization. To examine whether CREBH enhances HBV infection by affecting NTCP, we first examined changes in NTCP mRNA levels under CREBH overexpression. Full-length CREBH and truncated versions of CREBH-122 and CREBH-122-318 slightly downregulated NTCP mRNA levels, while other truncated versions did not alter NTCP mRNA transcription.

[0067] Example 4: CREBH knockdown inhibits hepatitis B virus infection

[0068] To test the necessity of CREBH in HBV infection, three CREBH-specific siRNAs were synthesized for knockdown experiments. Figure 6 As shown in A, all three siRNAs can effectively reduce the mRNA level of CREBH. In the corresponding HBV infection experiment, two NTCP-specific siRNAs were synthesized as positive controls. After HepG2-NTCP cells were transfected with CREBH siRNA, HBV infection was performed and HBeAg in the culture supernatant was detected (the results are shown in Figure 6 B) and HBsAg (results as shown Figure 6 C). The results show that knocking down CREBH can significantly affect HBV infection, and the degree of inhibition is comparable to the effect of knocking down NTCP on infection, indicating that CREBH is important in HBV infection.

[0069] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. SEQUENCE LISTING <110> Beijing Institute of Life Sciences <120> Specific host factors of hepatitis B virus infection and their applications <130> WO21551BSCN <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 461 <212> PRT <213> Homo sapiens <400> 1 Met Asn Thr Asp Leu Ala Ala Gly Lys Met Ala Ser Ala Ala Cys Ser 1 5 10 15 Met Asp Pro Ile Asp Ser Phe Glu Leu Leu Asp Leu Leu Phe Asp Arg 20 25 30 Gln Asp Gly Ile Leu Arg His Val Glu Leu Gly Glu Gly Trp Gly His 35 40 45 Val Lys Asp Gln Gln Val Leu Pro Asn Pro Asp Ser Asp Asp Phe Leu 50 55 60 Ser Ser Ile Leu Gly Ser Gly Asp Ser Leu Pro Ser Ser Pro Leu Trp 65 70 75 80 Ser Pro Glu Gly Ser Asp Ser Gly Ile Ser Glu Asp Leu Pro Ser Asp 85 90 95 Pro Gln Asp Thr Pro Pro Arg Ser Gly Pro Ala Thr Ser Pro Ala Gly 100 105 110 Cys His Pro Ala Gln Pro Gly Lys Gly Pro Cys Leu Ser Tyr His Pro 115 120 125 Gly Asn Ser Cys Ser Thr Thr Thr Pro Gly Pro Val Ile Gln Val Pro 130 135 140 Glu Ala Ser Val Thr Ile Asp Leu Glu Met Trp Ser Pro Gly Gly Arg 145 150 155 160 Ile Cys Ala Glu Lys Pro Ala Asp Pro Val Asp Leu Ser Pro Arg Cys 165 170 175 Asn Leu Thr Val Lys Asp Leu Leu Leu Ser Gly Ser Ser Gly Asp Leu 180 185 190 Gln Gln His His Leu Gly Ala Ser Tyr Leu Leu Arg Pro Gly Ala Gly 195 200 205 His Cys Gln Glu Leu Val Leu Thr Glu Asp Glu Lys Lys Leu Leu Ala 210 215 220 Lys Glu Gly Ile Thr Leu Pro Thr Gln Leu Pro Leu Thr Lys Tyr Glu 225 230 235 240 Glu Arg Val Leu Lys Lys Ile Arg Arg Lys Ile Arg Asn Lys Gln Ser 245 250 255 Ala Gln Glu Ser Arg Lys Lys Lys Lys Glu Tyr Ile Asp Gly Leu Glu 260 265 270 Thr Arg Met Ser Ala Cys Thr Ala Gln Asn Gln Glu Leu Gln Arg Lys 275 280 285 Val Leu His Leu Glu Lys Gln Asn Leu Ser Leu Leu Glu Gln Leu Lys 290 295 300 Lys Leu Gln Ala Ile Val Val Gln Ser Thr Ser Lys Ser Ala Gln Thr 305 310 315 320 Gly Thr Cys Val Ala Val Leu Leu Leu Ser Phe Ala Leu Ile Ile Leu 325 330 335 Pro Ser Ile Ser Pro Phe Gly Pro Asn Lys Thr Glu Ser Pro Gly Asp 340 345 350 Phe Ala Pro Val Arg Val Phe Ser Arg Thr Leu His Asn Asp Ala Ala 355 360 365 Ser Arg Val Ala Ala Asp Ala Val Pro Gly Ser Glu Ala Pro Gly Pro 370 375 380 Arg Pro Glu Ala Asp Thr Thr Arg Glu Glu Ser Pro Gly Ser Pro Gly 385 390 395 400 Ala Asp Trp Gly Phe Gln Asp Thr Ala Asn Leu Thr Asn Ser Thr Glu 405 410 415 Glu Leu Asp Asn Ala Thr Leu Val Leu Arg Asn Ala Thr Glu Gly Leu 420 425 430 Gly Gln Val Ala Leu Leu Asp Trp Val Ala Pro Gly Pro Ser Thr Gly 435 440 445 Ser Gly Arg Ala Gly Leu Glu Ala Ala Gly Asp Glu Leu 450 455 460 <210> 2 <211> 340 <212> PRT <213> Artificial Sequence <220> <223> CREBH-122 <400> 2 Met Cys Leu Ser Tyr His Pro Gly Asn Ser Cys Ser Thr Thr Thr Pro 1 5 10 15 Gly Pro Val Ile Gln Val Pro Glu Ala Ser Val Thr Ile Asp Leu Glu 20 25 30 Met Trp Ser Pro Gly Gly Arg Ile Cys Ala Glu Lys Pro Ala Asp Pro 35 40 45 Val Asp Leu Ser Pro Arg Cys Asn Leu Thr Val Lys Asp Leu Leu Leu 50 55 60 Ser Gly Ser Ser Gly Asp Leu Gln Gln His His Leu Gly Ala Ser Tyr 65 70 75 80 Leu Leu Arg Pro Gly Ala Gly His Cys Gln Glu Leu Val Leu Thr Glu 85 90 95 Asp Glu Lys Lys Leu Leu Ala Lys Glu Gly Ile Thr Leu Pro Thr Gln 100 105 110 Leu Pro Leu Thr Lys Tyr Glu Glu Arg Val Leu Lys Lys Ile Arg Arg 115 120 125 Lys Ile Arg Asn Lys Gln Ser Ala Gln Glu Ser Arg Lys Lys Lys Lys 130 135 140 Glu Tyr Ile Asp Gly Leu Glu Thr Arg Met Ser Ala Cys Thr Ala Gln 145 150 155 160 Asn Gln Glu Leu Gln Arg Lys Val Leu His Leu Glu Lys Gln Asn Leu 165 170 175 Ser Leu Leu Glu Gln Leu Lys Lys Leu Gln Ala Ile Val Val Gln Ser 180 185 190 Thr Ser Lys Ser Ala Gln Thr Gly Thr Cys Val Ala Val Leu Leu Leu 195 200 205 Ser Phe Ala Leu Ile Ile Leu Pro Ser Ile Ser Pro Phe Gly Pro Asn 210 215 220 Lys Thr Glu Ser Pro Gly Asp Phe Ala Pro Val Arg Val Phe Ser Arg 225 230 235 240 Thr Leu His Asn Asp Ala Ala Ser Arg Val Ala Ala Asp Ala Val Pro 245 250 255 Gly Ser Glu Ala Pro Gly Pro Arg Pro Glu Ala Asp Thr Thr Arg Glu 260 265 270 Glu Ser Pro Gly Ser Pro Gly Ala Asp Trp Gly Phe Gln Asp Thr Ala 275 280 285 Asn Leu Thr Asn Ser Thr Glu Glu Leu Asp Asn Ala Thr Leu Val Leu 290 295 300 Arg Asn Ala Thr Glu Gly Leu Gly Gln Val Ala Leu Leu Asp Trp Val 305 310 315 320 Ala Pro Gly Pro Ser Thr Gly Ser Gly Arg Ala Gly Leu Glu Ala Ala 325 330 335 Gly Asp Glu Leu 340 <210> 3 <211> 197 <212> PRT <213> Artificial Sequence <220> <223> CREBH-122-318 <400> 3 Pro Cys Leu Ser Tyr His Pro Gly Asn Ser Cys Ser Thr Thr Thr Pro 1 5 10 15 Gly Pro Val Ile Gln Val Pro Glu Ala Ser Val Thr Ile Asp Leu Glu 20 25 30 Met Trp Ser Pro Gly Gly Arg Ile Cys Ala Glu Lys Pro Ala Asp Pro 35 40 45 Val Asp Leu Ser Pro Arg Cys Asn Leu Thr Val Lys Asp Leu Leu Leu 50 55 60 Ser Gly Ser Ser Gly Asp Leu Gln Gln His His Leu Gly Ala Ser Tyr 65 70 75 80 Leu Leu Arg Pro Gly Ala Gly His Cys Gln Glu Leu Val Leu Thr Glu 85 90 95 Asp Glu Lys Lys Leu Leu Ala Lys Glu Gly Ile Thr Leu Pro Thr Gln 100 105 110 Leu Pro Leu Thr Lys Tyr Glu Glu Arg Val Leu Lys Lys Ile Arg Arg 115 120 125 Lys Ile Arg Asn Lys Gln Ser Ala Gln Glu Ser Arg Lys Lys Lys Lys 130 135 140 Glu Tyr Ile Asp Gly Leu Glu Thr Arg Met Ser Ala Cys Thr Ala Gln 145 150 155 160 Asn Gln Glu Leu Gln Arg Lys Val Leu His Leu Glu Lys Gln Asn Leu 165 170 175 Ser Leu Leu Glu Gln Leu Light Light Leu Gln Ala Ile Val Val Gln Ser 180 185 190 Thr See Light See Ala 195 <210> 4 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> CREBH-211-318 <400> 4 Gln Glu Leu Val Leu Thr Glu Asp Glu Lys Lys Leu Leu Ala Lys Glu 1 5 10 15 Gly Ile Thr Leu Pro Thr Gln Leu Pro Leu Thr Lys Tyr Glu Glu Arg 20 25 30 Val Leu Lys Lys Ile Arg Arg Lys Ile Arg Asn Lys Gln Ser Ala Gln 35 40 45 Glu Ser Arg Lys Lys Lys Glu Tyr Ile Asp Gly Leu Glu Thr Arg 50 55 60 Met Ser Ala Cys Thr Ala Gln Asn Gln Glu Leu Gln Arg Lys Val Leu 65 70 75 80 His Leu Glu Lys Gln Asn Leu Ser Leu Leu Glu Gln Leu Lys Lys Leu 85 90 95 Gln Ala Ile Val Val Gln Ser Thr Ser Lys Ser Ala 100 105 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siCREBH -1 <400> 5 gcugcuggaa agauggcuu 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siCREBH -2 <400> 6 gcuccuggau cucuguuu 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siCREBH -3 <400> 7 cccucuugga gcaacugaa 19 <210> 8 <211> 305 <212> PRT <213> Artificial Sequence <220> <223> CREBH-C <400> 8 Put Asn Thr Asp Leu Ala Ala Gly Lys Put Ala Sir Ala Ala Cys Sir 1 5 10 15 Put Asp Pro Ile Asp Ser Phe Glu Leu Leu Asp Leu Leu Phe Asp Arg 20 25 30 Gln Asp Gly Ile Leu Arg His Val Glu Leu Gly Glu Gly Trp Gly His 35 40 45 Val Lys Asp Gln Gln Val Leu Pro Asn Pro Asp Ser Asp Asp Phe Leu 50 55 60 Ser Ser Ile Leu Gly Ser Gly Asp Ser Leu Pro Ser Ser Pro Leu Trp 65 70 75 80 Ser Pro Glu Gly Ser Asp Ser Gly Ile Ser Glu Asp Leu Pro Ser Asp 85 90 95 Pro Gln Asp Thr Pro Pro Arg Ser Gly Pro Ala Thr Ser Pro Ala Gly 100 105 110 Cys His Pro Ala Gln Pro Gly Lys Gly Pro Cys Leu Ser Tyr His Pro 115 120 125 Gly Asn Ser Cys Ser Thr Thr Thr Pro Gly Pro Val Ile Gln Val Pro 130 135 140 Glu Ala Ser Val Thr Ile Asp Leu Glu Met Trp Ser Pro Gly Gly Arg 145 150 155 160 Ile Cys Ala Glu Lys Pro Ala Asp Pro Val Asp Leu Ser Pro Arg Cys 165 170 175 Asn Leu Thr Val Lys Asp Leu Leu Leu Ser Gly Ser Ser Gly Asp Leu 180 185 190 Gln Gln His His Leu Gly Ala Ser Tyr Leu Leu Arg Pro Gly Ala Gly 195 200 205 His Cys Gln Glu Leu Val Leu Thr Glu Asp Glu Lys Lys Leu Leu Ala 210 215 220 Lys Glu Gly Ile Thr Leu Pro Thr Gln Leu Pro Leu Thr Lys Tyr Glu 225 230 235 240 Glu Arg Val Leu Lys Lys Ile Arg Arg Lys Ile Arg Asn Lys Gln Ser 245 250 255 Ala Gln Glu Ser Arg Lys Lys Lys Lys Glu Tyr Ile Asp Gly Leu Glu 260 265 270 Thr Arg Met Ser Ala Cys Thr Ala Gln Asn Gln Glu Leu Gln Arg Lys 275 280 285 Val Leu His Leu Glu Lys Gln Asn Leu Ser Leu Leu Glu Gln Leu Lys 290 295 300 Lys 305 <210> 9 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> CREBH-307 <400> 9 Ala Ile Val Val Gln Ser Thr Ser Lys Ser Ala Gln Thr Gly Thr Cys 1 5 10 15 Val Ala Val Leu Leu Leu Ser Phe Ala Leu Ile Ile Leu Pro Ser Ile 20 25 30 Ser Pro Phe Gly Pro Asn Lys Thr Glu Ser Pro Gly Asp Phe Ala Pro 35 40 45 Val Arg Val Phe Ser Arg Thr Leu His Asn Asp Ala Ala Ser Arg Val 50 55 60 Ala Ala Asp Ala Val Pro Gly Ser Glu Ala Pro Gly Pro Arg Pro Glu 65 70 75 80 Ala Asp Thr Thr Arg Glu Glu Ser Pro Gly Ser Pro Gly Ala Asp Trp 85 90 95 Gly Phe Gln Asp Thr Ala Asn Leu Thr Asn Ser Thr Glu Glu Leu Asp 100 105 110 Asn Ala Thr Leu Val Leu Arg Asn Ala Thr Glu Gly Leu Gly Gln Val 115 120 125 Ala Leu Leu Asp Trp Val Ala Pro Gly Pro Ser Thr Gly Ser Gly Arg 130 135 140 Ala Gly Leu Glu Ala Ala Gly Asp Glu Leu 145 150 <210> 10 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> CREBH-211-C <400> 10 Gln Glu Leu Val Leu Thr Glu Asp Glu Lys Lys Leu Leu Ala Lys Glu 1 5 10 15 Gly Ile Thr Leu Pro Thr Gln Leu Pro Leu Thr Lys Tyr Glu Glu Arg 20 25 30 Val Leu Lys Lys Ile Arg Arg Lys Ile Arg Asn Lys Gln Ser Ala Gln 35 40 45 Glu Ser Arg Lys Lys Lys Lys Glu Tyr Ile Asp Gly Leu Glu Thr Arg 50 55 60 Met Ser Ala Cys Thr Ala Gln Asn Gln Glu Leu Gln Arg Lys Val Leu 65 70 75 80 His Leu Glu Lys Gln Asn Leu Ser Leu Leu Glu Gln Leu Lys Lys Leu 85 90 95 Gln Ala Ile Val Val Gln Ser Thr Ser Lys Ser Ala Gln Thr Gly Thr 100 105 110 Cys Val Ala Val Leu Leu Leu Ser Phe Ala Leu Ile Ile Leu Pro Ser 115 120 125 Ile Ser Pro Phe Gly Pro Asn Lys Thr Glu Ser Pro Gly Asp Phe Ala 130 135 140 Pro Val Arg Val Phe Ser Arg Thr Leu His Asn Asp Ala Ala Ser Arg 145 150 155 160 Val Ala Ala Asp Ala Val Pro Gly Ser Glu Ala Pro Gly Pro Arg Pro 165 170 175 Glu Ala Asp Thr Thr Arg Glu Glu Ser Pro Gly Ser Pro Gly Ala Asp 180 185 190 Trp Gly Phe Gln Asp Thr Ala Asn Leu Thr Asn Ser Thr Glu Glu Leu 195 200 205 Asp Asn Ala Thr Leu Val Leu Arg Asn Ala Thr Glu Gly Leu Gly Gln 210 215 220 Val Ala Leu Leu Asp Trp Val Ala Pro Gly Pro Ser Thr Gly Ser Gly 225 230 235 240 Arg Ala Gly Leu Glu Ala Ala Gly Asp Glu Leu 245 250

Claims

1. A truncated form of a specific host factor for hepatitis B virus infection, characterized in that: The amino acid sequence of the specific host factor for hepatitis B virus infection is shown in SEQ ID NO: 1, and the amino acid sequence of the truncated version is shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. An isolated nucleotide, characterized in that It encodes the truncated form described in claim 1.

3. A carrier, characterized in that The nucleotide according to claim 2 is inserted into the vector, and / or a truncated form of the specific host factor according to claim 1 can be exogenously expressed.

4. A cell into which the nucleotide according to claim 2 or the vector according to claim 3 has been exogenously introduced.

5. The cell according to claim 4, characterized in that The cells are selected from the group consisting of: HepG2 cells, HepG2-NTCP cells and human primary liver cells PHH.

6. The cell according to claim 5, characterized in that The cells are cultured in medium containing DMSO and / or insulin.

7. The cell according to claim 6, characterized in that The culture medium is PMM culture medium.

8. Use of the nucleotide according to claim 2 or the vector according to claim 3 in constructing a CREBH gene knockout non-human transgenic animal model, a CREBH gene knock-in non-human transgenic animal model, a hepatitis B virus-infected non-human transgenic animal model, or a hepatitis B virus-susceptible non-human transgenic animal model.

9. Use of the truncated form of claim 1, the nucleotide of claim 2, the vector of claim 3, the cell of any one of claims 4 to 7, or a non-human transgenic animal model constructed using the nucleotide of claim 2 or the vector of claim 3 in screening drugs for treating and / or preventing hepatitis B virus infection and / or diseases induced by hepatitis B virus infection.

10. A method for screening drugs for treating and / or preventing hepatitis B virus infection or diseases induced by hepatitis B virus infection, characterized in that: The method comprises: using the truncated form of claim 1, the nucleotide of claim 2, the vector of claim 3, the cell of any one of claims 4 to 7, or a non-human transgenic animal model constructed using the nucleotide of claim 2 or the vector of claim 3.

11. Use of a substance for preventing or reducing the transcription of the nucleotide sequence of claim 2 in the preparation of a medicament for treating and / or preventing hepatitis B virus infection and diseases induced by hepatitis B virus infection in mammals. in, The substance that prevents or reduces the transcription of the nucleotide according to claim 2 is an siRNA targeting the nucleotide, and the siRNA of the nucleotide is selected from the sequence shown in SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO:

7.

12. A drug for treating and / or preventing hepatitis B virus infection and diseases induced by hepatitis B virus infection in mammals, characterized in that: The drug comprises: the truncated form according to claim 1.

13. A drug for treating and / or preventing hepatitis B virus infection and diseases induced by hepatitis B virus infection in mammals, characterized in that: The active ingredient of the drug is: siRNA targeting the nucleotide according to claim 2, wherein the siRNA of the nucleotide is selected from the sequence shown in SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.

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