SPECIFIC HOST FACTOR OF HEPATITIS B VIRUS INFECTION, AND USE THEREOF

Abstract

Provided is a host factor specific for hepatitis B virus (HBV) infection. The specific host factor CREBH can remarkably enhance HBV infection. The specific host factor can, on the one hand, enhance entry of HBV, and on the other hand, enhance transcription of HBV to some extent. In the CREBH regulatory pathway there is a specific host factor SCARF2. During HBV infection, an N-terminus EGF-like domain of SCARF2 plays a crucial role in the infection and entry of HBV. The two correlated specific host factors provide a new target for inhibiting HBV infection.

Claims

1. A cell expressing a first exogenous host factor specific for hepatitis B virus (HBV) infection having an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 8, and/or a truncated form of the first exogenous host factor comprising an N-terminus domain of the first exogenous host factor.

2. The cell according to claim 1, wherein the truncated form of the first exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO: 9 or 10, or having an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 9 or 10.

3. The cell according to claim 1 further comprising a second exogenous host factor specific for HBV infection and/or a truncated form thereof capable of regulating expression or function of the first specific host factor, wherein the second exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO:1, or has an amino acid sequence having at least 90% identity to the amino acid sequence as shown in SEQ ID NO:1.

4. The cell according to claim 1, wherein the truncated form of the second exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

5. The cell according to claim 1, wherein the cell is selected from a group consisting of HepG2 cells, HepG2-NTCP cells, and primary human hepatocytes (PHHs).

6. The cell according to claim 1, wherein the cells are cultured in a medium containing DMSO and/or insulin.

7. The cell according to claim 6, wherein the medium is PMM medium.

8. A nucleotide molecule encoding a first host factor specific for hepatitis B virus (HBV) infection having an amino acid sequence as shown in SEQ ID NO:8 or having an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO:8, or a truncated form of the first host factor which comprises an N-terminus domain of the specific host factor.

9. The nucleotide molecule according to claim 8, wherein the truncated form of the first host factor comprises an amino acid sequence as shown in SEQ ID NO: 9 or 10, or having an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 9 or 10.

10. A method for screening a drug for treatment and/or prevention of HBV infection or related diseases by using a cell or an non-human animal, which comprises a first exogenous host factor specific for hepatitis B virus (HBV) infection having an amino acid sequence as shown in SEQ ID NO:8 or having an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO:8, and/or a truncated form of the first exogenous host factor comprising an N-terminus domain of the first exogenous host factor.

11. The method according to claim 10, wherein the truncated form of the first exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO: 9 or 10, or having an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 9 or 10.

12. The method according to claim 10, wherein the cell or an non-human animal further comprises a second exogenous host factor specific for HBV infection and/or a truncated form thereof capable of regulating expression or function of the first exogenous host factor, wherein the second exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO:1, or has an amino acid sequence having at least 90% identity to the amino acid sequence as shown in SEQ ID NO:1.

13. The method according to claim 10, wherein the truncated form of the second exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

14. The method according to claim 10, wherein the cell is selected from a group consisting of HepG2 cells, HepG2-NTCP cells, and primary human hepatocytes (PHHs).

15. The method according to claim 10, wherein the cells are cultured in a medium containing DMSO and/or insulin.

16. The method according to claim 15, wherein the medium is PMM medium.

17. The method according to claim 10, wherein the non-human animal is selected from a group consisting of: a non-human transgenic animal in which the first exogenous host factor-encoding gene is knock out, a non-human transgenic animal in which the first exogenous host factor-encoding gene is knock in, a non-human transgenic animal infected with HBV, or a non-human transgenic animal susceptible to HBV infection.

18. A drug for treatment and/or prevention of HBV infection and related diseases in mammals, comprising: a first exogenous host factor specific for hepatitis B virus (HBV) infection having an amino acid sequence as shown in SEQ ID NO: 8 or having an amino acid sequence that has at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 8, or a truncated form of the first exogenous host factor which comprises an N-terminus domain of the first exogenous host factor, a second exogenous host factor specific for HBV infection or a truncated form thereof capable of regulating expression or function of the first specific host factor, wherein the second exogenous host factor comprises an amino acid sequence as shown in SEQ ID NO:1, or has an amino acid sequence having at least 90% identity to the amino acid sequence as shown in SEQ ID NO:1, a nucleotide molecule encoding the first exogenous host factor, a substance capable of inhibiting or decreasing protein expression/function of the first exogenous host factor, and/or a substance capable of inhibiting or decreasing interaction between the first or second exogenous host factor and HBV.

19. The drug according to claim 18, wherein the drug is an siRNA molecule which comprises at least one nucleotide sequence selected from a group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 shows the results of screening of a cDNA library in Example 1.

[0051] FIG. 2 is a schematic diagram showing the construction of truncated clones of CREBH in Example 2.

[0052] FIGS. 3A, 3B, 3C, and 3D show the effects of truncated forms of CREBH on HBV infection in Example 2. (FIG. 3A) HbeAg level in infection in the presence of 1% PEG. (FIG. 3B) HbsAg level in infection in the presence of 1% PEG. (FIG. 3C) HbeAg level in infection in the presence of 5% PEG. (FIG. 3D) HBsAg level in infection in the presence of 5% PEG.

[0053] FIGS. 4A, 4B, and 4C show the results of infection at different viral titers after transduction of CREBH and truncated clones thereof in Example 2. (FIG. 4A) Results of infection in the presence of 1% PEG. (FIG. 4B) Results of infection in the presence of 5% PEG. (FIG. 4C) A schematic diagram of relative infection percentages. Four groups namely 1% PEG CREBH-122 group, 1% PEG PWPI group, 5% PEG PWPI group, 5% PEG CREBH-122 group were selected for experiments. For each group, HBeAg level on Day 6 post infection at 100% virus infection was calculated as 100%, and infection values at other viral titers each were divided by this 100% to obtain the percentages of the relative infection percentages.

[0054] FIGS. 5A, 5B, 5C, and 5D are schematic diagrams showing effects of transduction of CREBH and truncated forms thereof after HBV infection in Example 2. (FIG. 5A) HbeAg level in infection in the presence of 1% PEG. (FIG. 5B) HbsAg level in infection in the presence of 1% PEG. (FIG. 5C) HbeAg level in infection in the presence of 5% PEG. (FIG. 5D) HBsAg level in infection in the presence of 5% PEG.

[0055] FIGS. 6A, 6B, and 6C are schematic diagrams showing the effects of knockdown of CREBH on infection in Example 4. (FIG. 6A) Three CREBH siRNAs were transfected into HepG2-NTCP cells. At 48 hours after the transfection, RNA was collected using TRIzol, and relative expression of CREBH was measured by reverse transcription. (FIG. 6B) HBeAg level in supernatant after HBV infection. (FIG. 6C) HBsAg level in supernatant after HBV infection.

[0056] FIGS. 7A, 7B, 7C, 7D, 7E, and 7F are schematic diagrams showing the results of changes in expression of SCARF2 after transduction of CREBH and truncated forms thereof in Example 5. (FIGS. 7A-7E) Changes in relative expression of SCARF2 mRNA after transduction of CREBH, CREBH-122, CREBH-122-318, CREBH-211-318, and CREBH-307, respectively. (FIG. 7F) Statistics for relative expression of SCARF2 mRNA in each group.

[0057] FIGS. 8A, 8B, and 8C are schematic diagrams showing the effects of transduction of SCARF2 on infection in Example 6. (FIG. 8A) HepG2-NTCP cells were transduced with SCARF2, CREBH-122, or PWPI, and then infected with HBV in the presence of 1% PEG. (FIG. 8B) HepG2-NTCP cells were transduced with SCARF2, CREBH-122, or PWPI, and then infected with HBV in the presence of 5% PEG. (FIG. 8C) Nuclear and cytoplasmic fractions of the cells at 0 hour, 6 hours, 24 hours, and 72 hours after infection were isolated, and content of HBV DNA in each of the fractions was detected by southern-blot.

[0058] FIGS. 9A, 9B, and 9C are schematic diagrams showing the effects of knockdown of SCARF2 on infection in Example 6. (FIG. 9A) Five siRNAs of SCARF2 were designed and synthesized, and transfected into HepG2-NTCP cells. At 48 hours after the transfection, RNA was collected by TRIzol, and relative expression of SCARF2 was measured by reverse transcription. (FIG. 9B) Infection was conducted after SCARF2 was knocked down by siRNA. NTCP siRNA was used as a positive control. HbeAg in supernatant of the medium was detected. (FIG. 9C) Infection was conducted after SCARF2 was knocked down by siRNA. NTCP siRNA was used as a positive control. HBsAg in supernatant of the medium was detected.

[0059] FIGS. 10A, 10B, and 10C are schematic diagrams showing the results of analysis of functions of domains of SCARF2 in Example 6. (FIG. 10A) Schematic diagram showing structure of SCARF2 gene. (FIG. 10B) Results of N-terminal HA-tag staining after transducing AC12 cells with mutants with deletion of different EGF-like domains. (FIG. 10C) ELISA results of HBeAg levels in HBV infection occurred after transduction of different truncated forms. EGF4-6 was involved in change of localization of SCARF2 in cells and affected HBV infection.

[0060] FIGS. 11A, 11B, 11C, and 11D are schematic diagrams showing the effects of exogenously expressed EGF4-6 protein on HBV infection tested in Example 7. EGF truncated protein was incubated at a concentration of 20 μg/mL with HBV, and then HBV infection was conducted. Judging from HBeAg level (FIG. 11A) and HBsAg level (FIG. 11B) in supernatant after the infection, addition of the protein could significantly enhance HBV infection. (FIG. 11C) Infected cells were harvested for southern-blot detection of cccDNA. (FIG. 11D) Results of quantification of cccDNA in cells by cccDNA quantification.

[0061] FIGS. 12A and 12B are schematic diagrams showing the direct interaction between EGF4-6 and HBV detected by equilibrium density gradient centrifugation in Example 7. (FIG. 12A) After addition of EGF4-6 protein, the peak position of virus fractions shifted backwards by 2 fractions. (FIG. 12B) Equilibrium density gradient centrifugation was performed after incubating EGF4-6 with the virus. The dotted line represents HBsAg level, and the solid line represents DNA copy numbers of the virus. The lower chart shows results of quantification of the protein in each of the fractions by ImageJ software after detection of content of EGF-6 protein in corresponding fractions.

[0062] FIGS. 13A and 13B are schematic diagrams showing the results of interaction between EGF4-6 domains of SCARF2 and the amino acids 69-108 of the preS1 segment of HBV L protein in Example 7. (FIG. 13A) Results of specific binding of SCARF2 EGF4-6 protein to a polypeptide containing amino acids 69-108 of preS1 (peptide.sup.69-108). (FIG. 13B) Results of measurement of dissociation constant (K.sub.d) between the polypeptide and SCARF2 EGF4-6 protein.

[0063] FIGS. 14A and 14B are schematic diagrams showing the results of intracellular localization of SCARF2, NTCP, and HBV DNA during HBV infection in Example 8. (FIG. 14A) Interaction between NTCP and SCARF2 during HBV infection labeled by proximity ligation assay (PLA) (image on the left). PLA results obtained after HBV infection was blocked by using A14 antibody against HBV preS1 region (image on the right). HBV infection significantly increased the interaction between NTCP and SCARF2. (FIG. 14B) Subcellular localization of HBV DNA and SCARF2 protein in HepG2-NTCP cells at 48 hours (upper images) and 72 hours (lower images) after the cells were infected with HBV. HBV DNA in situ hybridization and SCARF2 protein immunofluorescence staining were performed. In the experiments of HBV infection, results of intracellular co-localization of the viral DNA and SCARF2 indicate that SCARF2 was involved in the process of HBV infection. Cell nuclei were stained with DAPI (A, B).

[0064] FIGS. 15A, 15B, 15C, and 15D are schematic diagrams showing the results of changes in localization of SCARF2 in the early stage of HBV infection in Example 7. (FIG. 15A) After the stable expression of SCARF2-GFP in HepG2-NTCP cells, the position of SCARF2-GFP in subcellular localization changed 48 hours after HBV infection or HBV infection was blocked by A14. (FIG. 15B) SCARF2-GFP was overexpressed in HepG2-NTCP cells. Intracellular localization of SCARF2 in the cells was indicated by GFP signal, and meanwhile a nuclear pore complex on the nuclear membrane was indicated by immunofluorescently stained Nup153. “Transection” represents that the microscope is focused on a section of the nucleus. “Top” represents that the microscope is focused on the top of the nucleus. (FIG. 15C) Photographs obtained after reconstruction of SCARF2-GFP and Nup153 signals on the nuclear membrane using a Nikon SIM super-resolution microscope. (FIG. 15D) Schematic diagram showing localization of SCARF2-GFP, the nuclear pore complex, and HBV DNA signals after simultaneous observation thereof using a super-resolution microscope.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0065] The following embodiments are intended to illustrate the present invention, rather than limiting the scope of the present invention.

Example 1: Construction and Screening of a cDNA Library for Hepatocyte Membrane Protein Genes

[0066] 1. Effects of PEG on Infection

[0067] HepG2-NTCP (AC12) is an existing cell line that can be efficiently infected with HBV. It is a single clone selected and obtained in accordance with stable expression of NTCP in HepG2 cancer cell line. However, according to existing studies and data known in the laboratory, efficient HBV infection of HepG2-NTCP cells still requires additional addition of PEG (PEG8000) as well as a viral titer much higher than that required for in vivo infection. It is generally believed that the role of PEG is to enhance the contact between the virus and receptors on the cell membrane surface; meanwhile, the high viral titer used suggests that in cells cultured in vitro there may be host factors limiting the infection or a lack of infection-dependent host factors. Therefore, use of the virus at a low titer and use of PEG at a reduced concentration in genetic screening for the infection may help to obtain not only factors that can increase interaction between the virus and receptors on the cell membrane surface, but also key host factors for entry of the virus.

[0068] Experiments were conducted to first detect the infection of HepG2-NTCP cells in the presence of different concentrations of PEG. Results show that the infection of the HepG2-NTCP cells relies on the concentration of PEG added during the infection. The presence of 1% PEG and 2% PEG can lead to enhanced HBV infection, which however is much lower than the level of the infection achieved in the presence of 5% PEG.

[0069] 2. Construction and Screening of a Library for Hepatocyte Membrane Proteins

[0070] The present invention consulted and utilized the Human Protein Atlas database for the definition and grouping of liver-specifically expressed genes. There were altogether 426 liver-specifically expressed genes. Because it was speculated that host-restrictive factors affecting the infection would be more likely to be located on the cell membrane or the endomembrane system, the present invention selected 114 membrane protein genes for cloning from the 426 genes by taking advantage of the annotated subcellular localization information. The membrane protein genes were cloned into a PWPI vector by enzyme digestion and ligation and then subjected to sequencing and verification, by way of which a target library containing 76 genes was successfully constructed.

[0071] The cDNA library was prepared by packaging lentivirus, with HepG2-NTCP cells expressing the cDNA clones being packaged therein. After 24 hours, infection was conducted with HBV at a same tier in the presence of 1% PEG. Culture supernatant was collected every 2 days, and was tested with HBeAg and HBsAg kits. As can be seen from results shown in FIG. 1, clone No. 15 (CREBH) remarkably enhances HBV infection, and the HbeAg level is similar to that achieved in the infection in the presence of 5% PEG. Results of HBcA staining show that CREBH remarkably increases the number of HBcAg-positive cells, indicating that CREBH may enhance the infection efficiency.

Example 2: CREBH Versus HBV Infection

[0072] 1. Expression Pattern of CREBH in Tissues

[0073] Clone No. 15 encoded CREBH (Cyclic AMP Response Element-binding Protein H, CREB3L3) gene. Referring to GTEx database for information of CREBH gene, CREBH is specifically expressed in liver and small intestine tissues, but its expression level in liver is much higher than that in small intestine tissues.

[0074] The expression levels of CREBH in HepG2-NTCP cells, mPHH (humanized mouse derived primary hepatocyte) cells, liver cancer tissues and adjacent normal tissues of patients were simultaneously measured in the experiments. The expression levels of CREBH in HepG2-NTCP cells and mPHH cells are close to each other but not high. The expression levels of CREBH in liver cancer tissues and adjacent normal tissues are significantly higher than those in the cell lines, and the expression level in liver cancer tissues is slightly higher than that in the adjacent normal tissues. This indicates that the expression of CREBH is inhibited in the cell lines cultured in vitro, which may be one of the reasons why in vitro infection efficiency is lower than in vivo infection efficiency.

[0075] 2. Analysis of Functions of Truncated Forms of CREBH

[0076] Human CREBH consists of 461 amino acids and belongs to type II transmembrane protein. CREBH protein has, at its N-terminus, a bZIP domain that can bind to DNA and have a transmembrane region that can attach the protein to the endoplasmic reticulum membrane. Upon stimulated by a signal, CREBH can be transported to the Golgi membrane, where it is cleaved by Sire 1 Protease and Sire 2 Protease to release the N-terminus with a transcriptional activation function into the nucleus. Functions of the residual C-terminus are unknown. When it is comes to its regulatory functions, CREBH is often reported to be associated with lipid metabolism and cholesterol metabolism. Since the subcellular localization of domains at the two termini of CREBH are different, it is necessary to further detect which of the domains has the effect of enhancing HBV infection.

[0077] 3. Results of CREBH Secondary Structure Prediction

[0078] Secondary structure of CREBH was predicted through experiments using the CFSSP (Chou and Fasman Secondary Structure Prediction server) database. In accordance with results of the prediction, 3 breakpoints were selected from the middle part of CREBH protein to construct altogether 6 truncated clones, as shown in FIG. 2.

[0079] Results of the secondary structure prediction show that within amino acids 1-122 there is a segment of secondary structure with a low score, which may be an intrinsic disorder region. Such a region in transcription factors is usually closely related to transcriptional activation activity. In order to maintain integrity of the bZIP domain as much as possible, the second breakpoint was selected at amino acid 211, and amino acid 318 near the transmembrane region was selected as the third breakpoint. A total of 6 truncated form clones of CREBH were constructed. For each of the clones, an HA tag was added to its N-terminus and a c-myc tag was added to its C-terminus.

[0080] 4. Cellular Localization of Truncated Forms of CREBH

[0081] In order to study the localization of CREBH in HepG2 cell line, an HA tag was attached to the N-terminus of each of the CREBH and truncated forms thereof constructed above, and a c-myc tag was attached to the C-terminus thereof. HepG2-NTCP cells were transduced with lentivirus. The HA tag and the c-myc tag were stained to observe the localization of different truncated forms of CREBH in the cells.

[0082] Results of the staining show that in full-length clone of CREBH, the color on the N-terminus and the color on the C-terminus are slightly separated, with a small part of the N-terminus of CREBH being localized to the nucleus, while most of it remaining in the cytoplasm and overlapping with the color on the C-terminus. In the truncated clone CREBH-122, separation between the nuclear-localized N-terminus and the cytoplasmic-localized C-terminus is more apparent. This indicates that compared with the full-length CREBH, the N-terminus truncated CREBH-122 can be cleaved more efficiently, or the N-terminus domain of CREBH-122 is more stable in the nucleus. CREBH-C, CREBH-122-318, and CREBH-211-318 are localized in a similar pattern, i.e., being localized predominantly to the nucleus, while the shorter form CREBH-307 and CREBH-211-C lose their nuclear localization. This indicates that the nuclear localization signal of CREBH may be located within amino acids 211-318.

[0083] In order to detect whether each truncated form still had transcriptional activation ability, experiments were conducted by using the activation of APOA4 gene reported in the literature as an indicator of the transcriptional activation ability of CREBH. After the truncated forms were transduced into HepG2-NTCP cells, relative expression of APOA4 was detected. It was then found that the full-length CREBH had the strongest activation effect on APOA4 gene. CREBH-C also had relatively high activation efficiency, which however was about ⅓ of the activation efficiency of the full-length CREBH. Although the N-terminus of CREBH-C was the same as that of the full-length CREBH, the C-terminus of CREBH-C did not have the transmembrane region on the part that had been cleaved; it might be possible that it was this small region that affected the stability of the protein, thereby indirectly reducing the ability of CREBH-C to activate APOA4. CREBH-122 and CREBH-122-318 were also capable of activating APOA4 gene to some extent, but were much less capable than the full-length CREBH and CREBH-C, indicating that the part of the domain of CREBH capable of transcriptional activation was located within amino acids 1-122.

[0084] 5. Roles of Truncated Forms of CREBH in HBV Infection

[0085] HepG2-NTCP cells were transduced with lentivirus embedded with CREBH and truncated forms thereof. After 24 hours, the cells were re-seeded in a culture plate, and the culture medium was changed to PMM. After 24 hours, the cells were infected with HBV in the presence of 1% PEG and 5% PEG respectively. Supernatant was collected every 2 days and assayed using an ELISA kit. Results of the infection are shown in FIGS. 3A-D. As can be seen from the results, compared with the PWPI-B empty control, the full-length CREBH, CREBH-122, CREBH-C, CREBH-122-318, and CREBH-211-C can all increase the infection efficiency in the presence of 1% PEG and 5% PEG. Among them, CREBH-122 exhibits an enhancement effect significantly higher than that of other experimental groups.

[0086] HBcAg staining was also performed in the experiments, and results were consistent with the ELISA results. In addition to the enhancement effects of CREBH as well as its truncated clones CREBH-122 and CREBH-122-318 on HBV infection, it was also observed that CREBH-307 and CREBH-211-318 to some certain extent inhibited the infection, which was consistent with the ELISA results. It was thus speculated that these two truncated forms might have exerted a dominant-negative effect.

[0087] In short, the enhancement effect of CREBH on HBV infection depends on its own transcriptional activation ability, and CREBH-122 can significantly increase HBV antigens and the number of stained HBcAg positive cells. For this reason, CREBH-122, instead of full-length CREBH, was used to study its specific mechanism of action in subsequent experiments.

[0088] 6. Enhancement Effects of CREBH-122 on Infection at Different Viral Titers

[0089] In addition to being affected by PEG, in vitro HBV infection also relies on the titer of the virus used. Therefore, in experiments, CREBH-122 and CREBH-122-318 which had the strongest enhancement effects, as well as the truncated clone CREBH-211-318 which had a slightly inhibitory effect, were used to be in comparison to a PWPI empty control. Differences in infection efficiency at different viral titers were tested. If the infection occurred under completely ideal conditions, the index of the virus infection would be in a single-factor linear relationship with the amount of the virus used. If there were one or more other limiting factors, then the curve for the infection would fall out of linear correlation and the infection would decrease significantly with the decrease of the viral titer.

[0090] Specifically, HepG2-NTCP cells were transduced with lentivirus embedded with CREBH and truncated forms thereof. After 24 hours, the cells were reseeded in a culture plate, and cultured for 24 hours with the culture medium having been changed to PMM. With 160 ul virus/200 ul system under normal infection being calculated as a 100% viral titer, the virus was added to the infection system at a 50% titer, 25% titer, and 12.5% titer, and PMM was added to the volume. Infection with diluted HBV was performed in the presence of 1% PEG and 5% PEG, respectively. ELISA results of the infection show that CREBH and truncated forms thereof had the same effects on the infection at different viral tiers.

[0091] As can be seen from the results in FIG. 4A, in the presence of 1% PEG, the infection in the truncated form groups or the control group is reduced significantly with the decrease of the viral titer. As can be seen from the results in FIG. 4B, in the presence of 5% PEG, the reduction of the infection in cells transduced with CREBH-122 and CREBH-122-318 with the decrease of the viral titer is moderated.

[0092] In order to demonstrate the results more clearly, HBeAg levels on Day 6 day after infection in the CREBH-122 group and the PWPI empty group were selected and used in the experiments for charting. For each group, HBeAg level at 100% virus infection was calculated as 100%, and infection values at other viral titers each were divided by this 100% to obtain percentages of the relative infection values (infection percentages). A line graph was drawn by taking the infection percentage as the ordinate and the viral titer as the abscissa, and results are shown in FIG. 4C. As can be seen from the figure, the curves obtained of the infection in the 1% PEG CREBH-122 group, the 1% PEG PWPI group, and the 5% PEG PWPI group are similar. Transduction of CREBH-122 in the presence of 5% PEG, however, remarkably renders the curve closer to be linear. Pearson r values were calculated and a higher correlation was seen in the 5% PEG CREBH-122 group (1% PEG CREBH-122=0.986, 1% PEG PWPI=0.968, 5% PEG=0.986, 5% PEG CREBH-122=0.994). This indicates that the transduction of CREBH-122 helped to overcome an important limiting factor for HBV infection of HepG2-NTCP.

[0093] 7. Roles of CREBH after HBV Infection

[0094] Increases in secretion of HBeAg and HBsAg after HBV infection may be caused by enhanced transcription of HBV cccDNA or enhanced protein secretion pathway, and such increases cannot directly reflect enhancement in virus entry. Besides, CREBH itself has a transcriptional activation function. It is therefore necessary to detect in which specific step of the HBV infection CREBH exerts the enhancement effect.

[0095] As can be seen from the results shown in FIGS. 5A-D, first, in the experiments, CREBH and its truncated clones were transduced into the HepG2-NTCP cell line having been infected with HBV. Effects of CREBH and truncated forms thereof on HBeAg and HBsAg were consistent with those before the infection, indicating that CREBH might have indeed directly acted on HBV cccDNA itself. Second, HepG2-NTCP cells were transduced with letivirus at 24 hours after the infection with HBV. Supernatant was collected every 2 days to measure HBeAg and HBsAg. Compared with their enhancement effects on the infection in the presence of 1% PEG, the enhancement effects of CREBH and truncated forms thereof were stronger on the infection in the presence of 5% PEG. This was likely because when the infection occurred in the presence of 5% PEG, more cccDNA were formed in the cells as templates for transcription. This indirectly demonstrates that the effect of CREBH is stronger, and further proves that CREBH can enhance the transcription of HBV.

Example 3: CREBH Affects HBV Infection without Altering NTCP

[0096] NTCP is a key receptor in HBV infection and can directly bind to the PreS1 region on the virus. Many factors capable of affecting HBV infection may indirectly affect HBV infection by affecting the expression level or cellular localization of NTCP. In order to detect whether CREBH enhances HBV infection by affecting NTCP, changes in NTCP mRNA level under the condition of overexpressing CREBH was first detected. Full-length CREBH as well as truncated forms CREBH-122 and CREBH-122-318 slightly down-regulated the NTCP mRNA level, and other truncated forms did not change the transcription of NTCP mRNA.

Example 4: CREBH Knockdown Inhibits HBV Infection

[0097] To test necessity of CREBH in HBV infection, three CREBH-specific siRNAs were synthesized for knockdown experiments. qPCR results are shown in FIG. 6A. All the three siRNAs can effectively reduce CREBH mRNA level. In corresponding HBV infection experiments, two NTCP-specific siRNAs were synthesized for positive controls. After the CREBH siRNAs and the NTCP-specific siRNAs were separately transfected into HepG2-NTCP cells, HBV infection was performed. HBeAg and HBsAg in culture supernatant were detected respectively (results are shown in FIG. 6B and FIG. 6C respectively). As can be seen from the results, knockdown of CREBH can significantly affect and inhibit HBV infection to a degree that is comparable to the effect of knockdown of NTCP on the infection, which indicate that CREBH is important in HBV infection.

Example 5: Identification and Cloning of a CREBH-Regulated Host Factor

[0098] 1. Transcriptome Sequencing Analysis of Truncated Forms of CREBH

[0099] Since the results of the infection occurred after the transduction of truncated forms of CREBH indicate that the transcriptional activation function of CREBH is necessary for enhancement of the infection, and the shortest functional truncated form CREBH-122-318 is localized on the nucleus, it is inferred that it is by activating a downstream gene that CREBH enhances HBV entry. In order to more accurately determine the downstream major gene, HepG2-NTCP cells transduced with CREBH, CREBH-122, CREBH-307, CREBH-122-318, CREBH-211-318, PWPI-B, and PMM were selected and used for transcriptome sequencing experiments. Meanwhile, CREBH siRNA-2 was selected and used to knock down CREBH in HepG2-NTCP cells. siNC was used as a negative control. Changes in transcriptomes were detected. Because a primary one of the known functions of CREBH is transcriptional activation, not transcriptional repression, genes that were up-regulated after the transduction as compared to the control group as well as genes that were down-regulated after CREBH knockdown as compared to the control group were analyzed.

[0100] Analysis results show that transcriptomes of the cells transduced with the full-length CREBH and the functional truncated forms CREBH-122 and CREBH-122-318 are similar to each other, while transcriptome characteristics of the cells transduced with the dominant negative CREBH-307 and CREBH-211-318 are similar to those of the cells in which CREBH is knocked down. This result is similar to the role of truncated clones in the infection, and it further confirms the location of the downstream major gene. Then, 62 overlapping genes that were up-regulated by 2-fold in the CREBH/PWPI group and down-regulated by more than 1.2-fold in the CREBH knockdown experiment were cloned and analyzed, by way of which SCARF2 gene was finally identified. Functions of SCARF2 gene were verified in later experiments.

[0101] 2. SCARF2 is Regulated by CREBH and Truncated Clones Thereof

[0102] In order to more clearly demonstrate changes in expression of SCARF2 in cells transduced with CREBH and truncated forms thereof, corresponding mRNA sequencing data were displayed and analyzed by volcano plots. As shown in FIGS. 7A-F, CREBH can up-regulate the expression of SCARF2 gene, but not to a very significant level (p<0.01). However, the truncated forms CREBH-122 and CREBH-122-318, which have stronger enhancement effects on HBV infection, however, significantly up-regulates SCARF2. In contrast, the transduction with the truncated forms CREBH-211-318 and CREBH-307, which are incapable of HBV infection enhancement, does not lead to a change in SCARF2 expression. The same conclusion was also made from subsequent experiments using quantitative PCR, and the expression levels of SCARF2 were completely consistent with the enhancement effects of CREBH and its various truncated forms on HBV infection observed in the previous text. This indicates that SCARF2 may be a major gene for enhancement of HBV infection by CREBH.

Example 6: Effects of SCARF2 on Infection

[0103] 1. SCARF2 Significantly Enhances HBV Infection

[0104] In the present invention, SCARF2 was first overexpressed in HepG2-NTCP cell line to test the effects of SCARF2 on HBV infection. Results show that SCARF2 can enhance HBV infection in the presence of either 1% PEG or 5% PEG, with the enhancement effect being comparable to that of CREBH-122. Meanwhile, from HepG2-NTCP cell line stably expressing SCARF2, HBV DNA was isolated for analysis from the cytoplasm and the nucleus at different time points of HBV infection. Results are shown in FIGS. 8A-C and indicate that HBV DNA present in fractions of the cytoplasm of the cell expressing SCARF2 gene is slightly increased, while rcDNA and cccDNA present in fractions of the nucleus at a corresponding time point are detected to be significantly increased.

[0105] 2. Knockdown of SCARF2 Inhibits HBV Infection

[0106] 6 siRNAs were first synthesized to knock down SCARF2 in experiments. All the siRNAs could effectively reduce SCARF2 mRNA level. On this basis, HepG2-NTCP cells in which SCARF2 was knocked down were infected with HBV Results of the infection are shown in FIGS. 9A-C. HBV infection was effectively reduced in all the experimental groups with SCARF2 knockdown, with a degree of the reduction being similar to that of the reduction of the infection caused by NTCP knockdown. This indicates the importance of expression of SCARF2 gene in HBV infection.

[0107] 3. Analysis of Structure and Functional Domains of SCARF2

[0108] SCARF2 gene is a type I transmembrane protein. SCARF2 gene can be structurally divided into four parts: signal peptides, an N-terminus domain containing 7 EGF-like domains, a transmembrane region, and a C-terminus domain containing a Proline-rich domain. As shown in FIG. 10A, from the topological structure of SCARF2, the N-terminus containing the EGF-like domains is located on the inner side of the lumen or the outer side of the cell membrane, and therefore the N-terminus domains are likely to be directly correlated to HBV during HBV infection. In order to find out whether the N-terminus EGF-like domains contained a crucial segment, mutant clones each with a different one of the 7 EGF-like domains deleted were constructed. EGF2 and EGF3, as well as EGF5 and EGF6 contained parts that overlapped each other, and therefore these parts that could not be completely separated were temporarily taken as a whole in constructing the deletion mutants.

[0109] First, localization of each mutant of SCARF2 in a cell was determined by staining. Results are shown in FIG. 10B. Wild-type SCARF2 is localized to the cell membrane and cytoplasm; the EGF4 deletion mutant and the EGF5-6 deletion mutant are mainly localized to the cell membrane; and other mutants partially or completely lose their localization to the cell membrane. Consistent with results of HBV infection of corresponding mutants-overexpressed cell lines (FIG. 10C), overexpression of the two mutants with significantly increased cell membrane localization does not alter HBV infection efficiency, whereas the mutants with other EGFs deleted have enhancement effects on the infection that are comparable to the effect of wild-type SCARF2. It is thus proved that EGF4-6 of SCARF2 may be necessary for enhancing HBV infection, and that SCARF2 may function in the intracellular membrane system rather than on the cell membrane surface.

Example 7: Interaction of SCARF2 with HBV

[0110] 1. Expression and Purification of Extracellular EGF Domains of SCARF2

[0111] In order to examine whether the N-terminus of SCARF2 could directly interact with HBV, in the present invention, the N-terminus of different N-terminus domain truncated forms of SCARF2 was labeled with an HA tag, and the C-terminus thereof was fused with a human Fc tag (IgG1), with a Precision tag being introduced between the two. The fusion protein was cloned into pCAGGs vector, and the plasmid was transfected into 293F cells and purified using Protein A Beads.

[0112] 2. Effects of Truncated Proteins of SCARF2 on HBV Infection

[0113] In order to examine whether there was direct interaction between dSCARF2 proteins and HBV, in the experiment, proteins purified in vitro each were co-incubated at a concentration of 20 μg/mL with the virus, and HBV infection was conducted after the incubation. Early interaction between a virus and a soluble expressed nonfunctional receptor protein usually leads to loss and reduction of infectivity, and this method is also usually used to examine interaction between a protein and a virus. However, co-incubation of SCARF2 proteins with the virus could enhance the infection to some degree, and EGF4-6 could significantly enhance the infection (results are shown in FIGS. 11A-D). The results to some extent prove the interaction between the EGF-like domains of SCARF2 between the virus, and also indicate that the mode of action of SCARF2 is different from that of traditional viral receptors. It is known that soluble SCARF2 exists in blood, and soluble SCARF2 may have an enhancement effect on HBV infection.

[0114] 3. Verification of Binding of SCARF2 Proteins to HBV by Equilibrium Density Gradient Centrifugation

[0115] Binding of SCARF2 to the virus was also verified in the experiment by equilibrium density gradient centrifugation. In equilibrium density gradient centrifugation, Nycodenz® was used as a medium, and different fractions of the virus were separated according to different densities thereof. In the experiment, the virus used was HBV produced by transfection of Huh7 cells. The virus produced as such contained a large amount of naked nucleocapsid fractions not enveloped by the viral envelope but having HBV DNA. These fractions, compared with completely enveloped virus particles, had a higher density, and therefore could be detected by qPCR detection for HBV DNA after being ultra-centrifugated so as to be separated from the later.

[0116] In the experiment, the virus was first incubated with SCARF2 EGF4-6 proteins and then centrifuged. Compared with the negative control without addition of the proteins, the addition of the two proteins caused the position of the virus particles to be shifted back by 2 fractions. This suggests that the binding of the proteins results in a change in the density of the viral particles and thus a change in the position of the viral particles in equilibrium density gradient centrifugation (results are shown in FIG. 12A).

[0117] Meanwhile, because the proteins added contained the HA tags, it was possible to detect SCARF2 EGF4-6 proteins contained in each of the fractions. After co-incubating of EGF4-6 with the virus, the fractions obtained by ultracentrifugation were subjected to a dot blot experiment (results are shown in FIG. 12B), and the content of EGF4-6 proteins in each of the fractions was measured by using an anti-HA antibody. Results were quantified by ImageJ and are shown in FIG. 12B (the lower chart). Compared with the control group without the virus, the co-incubating of EGF4-6 with the virus leads to detection of presence of the proteins in fractions 9-11, which position is consistent with the position (fractions 9-11) of intact particles of the virus but inconsistent with the position (fractions 7-10) of subviral particles (SVPs). This further verifies the interaction of the extracellular EGF domains of SCARF2 protein with HBV.

[0118] 4. EGF4-6 Domains of SCARF2 Interact with Amino Acids 69-108 of the preS1 Segment of HBV L Protein

[0119] Compared with SVPs, the virus particles were rich in L protein, and the main functional segment of L protein was the preS1 segment; it was therefore speculated that the SCARF2 EGF4-6 proteins interact with the preS1 segment of the L protein of the virus. In order to further prove protein binding sites, three partially overlapping polypeptides (peptide.sup.2-48, peptide.sup.39-78, and peptide.sup.69-108) were first synthesized to cover the entire preS1 segment of the L protein of the virus. The C-terminus of each of the synthesized peptides was biotin labeled. It was found first by immunoprecipitation that the SCARF2 EGF4-6 proteins specifically bound to the polypeptide containing amino acids 69-108 of preS1 (peptide.sup.69-108) (results are shown in FIG. 13A). Then, the binding force between the polypeptide and the SCARF2 EGF4-6 proteins was measured using the ForteBio Octet system, with dissociation constant (K.sub.d) being determined to be 58 nM (results are shown in FIG. 13B). This experiment reveals specific, strong binding interaction between the SCARF2 protein and the virus.

Example 8: Mechanism of Action of SCARF2 in HBV Infection

[0120] 1. HBV Infection Promotes Mutual Approach of SCARF2 and NTCP

[0121] Proximity ligation assay (PLA) is one of commonly used methods for detection of protein-protein interaction in living cells. Because HBV bound to its receptor NTCP during the infection, and experiments had proved the interaction between SCARF2 and HBV, it was therefore speculated that in the early stage of HBV infection, HBV entry would cause NTCP and SCARF2 to approach each other. The interaction between NTCP and SCARF2 at 48 hours of HBV infection was detected by PLA assay.

[0122] FIGS. 14A-B are schematic diagrams showing the results of intracellular localization of SCARF2, NTCP, and HBV DNA during HBV infection. FIG. 14A shows the interaction between NTCP and SCARF2 during HBV infection labeled by proximity ligation assay (image on the left), and results of PLA after the infection was blocked using A14 antibody against the HBV preS1 region (image on the right). HBV infection significantly increased the interaction between NTCP and SCARF2. FIG. 14B shows subcellular localization of HBV DNA and SCARF2 protein in HepG2-NTCP cells at 48 hours (upper images) and 72 hours (lower images) after the cells were infected with HBV. HBV DNA in situ hybridization and SCARF2 protein immunofluorescence staining were performed. In experiments of the HBV infection, results of intracellular co-localization of the viral DNA and SCARF2 indicated that SCARF2 was involved in the process of HBV infection. Cell nuclei were stained with DAPI (A, B). As can be seen from the results, compared with the infection blocked group with addition of A14 antibody, the infection significantly increased the interaction between NTCP and SCARF2. In addition, co-localization of SCARF2 and the viral DNA was also found in the early stage of the infection by using HBV DNA in situ hybridization.

[0123] 2. SCARF2 is Involved in Transportation of the Virus to the Nuclear Pore Complex

[0124] To further understand the mechanism of action of SCARF2 in the process of HBV infection, a cell line stably expressing SCARF2-GFP protein was constructed using HepG2-NTCP cells. The localization of SCARF2 protein in the cells at 48 hours after HBV infection was observed by the fluorescence signal of GFP. As shown in FIG. 15A, compared with the infection blocked group with addition of the HBV A14 neutralizing antibody, the localization of SCARF2-GFP to the nuclear membrane was significantly increased at 48 hours after HBV infection. To further demonstrate the localization of SCARF2 to the nuclear membrane, HepG2-NTCP cells were transfected with SCARF2-GFP and cultured in PMM for 5 days to observe a positional relationship between SCARF2-GFP and the nuclear pore complex indicated by Nup153. As shown in FIG. 15B, co-localization of SCARF2-GFP and Nup153 to the nuclear membrane is observed from a section of the nucleus, and meanwhile the co-localization of SCARF2-GFP and the nuclear pore complex indicated by Nup153 can also be observed when focusing the microscope on the top of the nucleus. In the experiment, this structure was also reconstructed by using an N-SIM S super-resolution microscope. As can be seen from results shown in FIG. 15C, SCARF2-GFP is partially localized to a side of the cytoplasmic of the nuclear pore complex. At 48 hours after HBV infection, SCARF2-GFP, the nuclear pore complex, and HBV DNA signals were simultaneously observed using a super-resolution microscope, which clearly showed the co-localization of the three (results are shown in FIG. 15D). The above experimental results suggest that SCARF2 may have mediated the transport of the HBV particles to the nuclear pore complex during the entry of the virus particles.

[0125] Although the present invention has been described in detail above by way of general description, specific embodiments and tests, it is obvious to those skilled in the art that modifications or improvements can be made thereto on the basis of the present invention. Such modifications or improvements made without departing from the spirit of the present invention all fall within the protection scope of the present invention.