Identification of glycogen synthase kinase 3alpha/beta as a host factor required for HBV transcription using high-throughput screening.

INTRODUCTION
HBV infection causes severe liver diseases, including chronic hepatitis, liver cirrhosis, and HCC.1 Although therapy with nucleos(t)ide analogs suppresses HBV replication, long-term therapy is needed to maintain low levels of HBV DNA. The covalently closed circular DNA (cccDNA) of HBV cannot be eradicated with current treatments. Therefore, the discovery of new therapeutic targets for patients with HBV is required for the development of anti-HBV therapies.
HBV belongs to the Hepadnaviridae family and contains a 3.2-kb partially double-stranded circular DNA (relaxed circular DNA).2 HBV attaches to heparin sulfate proteoglycans, such as glypican-5,3 and enters human hepatocytes through sodium taurocholate cotransporting polypeptide (NTCP).4 Then, the relaxed circular DNA is imported into the nucleus where it is repaired into cccDNA by host DNA repair enzymes, including tyrosyl-DNA-phosphodiesterase 2,5 DNA polymerase K,6 flap endonuclease 1,7 proliferating cell nuclear antigen, replication factor C complex, DNA polymerase δ,8 DNA ligase 1,9 and poly(ADP-ribose) polymerase 1.10 Viral gene expression from cccDNA is regulated by 4 promoter elements and 2 enhancer elements that transcribe a 3.5-kb pregenomic RNA (pgRNA) and 2.4, 2.1, and 0.7 kb RNAs.11 Host transcription factors, such as hepatocyte nuclear factor 1, 3, and 4 alpha, play important roles in the transcription of viral RNAs.12–14 pgRNA is reverse transcribed to generate viral cDNAs that are enveloped and secreted as infectious virions. Identification of NTCP as a functional receptor for HBV has led to the development of cell culture infection systems for studying HBV replication. HBV genome replication-tropic cell lines, such as HepG2 and Huh7 cells, expressing NTCP provide a good opportunity to clarify the HBV life cycle. However, to understand the precise molecular events involved in the production of infectious virions, unknown events of the viral life cycle must be clarified.
In this study, we established a system to monitor the HBV life cycle from transcription to egress using a recombinant HBV genome in which the gene encoding NanoLuc was incorporated. Because this system is easy to handle and is not time-consuming compared with other methods that evaluate virus production, we used it to screen anti-HBV candidates.
We performed high-throughput screening with a compound library of 1827 FDA-approved drugs. We identified known inhibitors of GSK3alpha/beta, namely, AZD1080, CHIR-98014, CHIR-98021, and BIO, as anti-HBV drugs. A detailed analysis revealed that GSK3alpha/beta plays an important role in HBV transcription through FOXK2 phosphorylation.

METHODS

Cells
HepG2-NTCP cells were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS: Cytiva), 100 U/mL penicillin, 100 μg/mL streptomycin (Nacalai Tesque), and 100 U/mL nonessential amino acids (Thermo Fisher Scientific). HepAD38 cells15 were maintained in DMEM/F-12 (Thermo Fisher Scientific) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 500 μg/mL G418 (Nacalai Tesque), 18 μg/mL hydrocortisone (Sigma), and 400 ng/mL tetracycline (TaKaRa Bio). Primary human hepatocytes (PHHs) were purchased from PhoenixBio and cultured according to the manufacturer’s protocols. To establish the GSK3alpha and GSK3beta double-knockout cell line, cells were transfected with GSK3alpha CRISPR plasmid and GSK3beta CRISPR plasmid (Santa Cruz Biotechnology) along with pcDNA3.1 Hygro(+). After transfection, the cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 U/mL nonessential amino acids, and 100 μg/mL hygromycin (Nacalai Tesque) for 3 weeks. Single colonies were collected, and gene deficiency was confirmed through western blotting. To generate the FOXK1 and FOXK2 double-knockout cell line, cells were transfected with pLV[CRISPR]-hCas9-T2A-Hygro-U6-hFOXK1 and pLV[CRISPR]-hCas9-T2A-Hygro-U6-hFOXK2 (VectorBuilder). After transfection, the cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 U/mL nonessential amino acids, and 100 μg/mL hygromycin for 3 weeks. Single colonies were collected, and gene deficiency was confirmed through western blotting.

Preparation of HBV
HBV genotype D was derived from HepAD38 cells cultured without tetracycline. Culture supernatant was collected every 3–4 days for 30 days after induction and passed through a 0.45-μm filter. HBV was precipitated with 13% PEG8000 containing 0.75 M NaCl and 2 mM HEPES [pH 7.6] and purified through precipitation with 20% sucrose in TNE buffer (10 mM Tris [pH 7.6], 50 mM NaCl, and 1 mM EDTA) at 100,000g for 3 hours. Viruses were suspended in Opti-MEM (Thermo Fisher Scientific) and stored at −80 °C until use.

HBV infection
HepG2-NTCP cells or PHHs were infected with HBV at 5000 or 100 genome equivalents (GEq)/cell, respectively, in the presence of 2% DMSO and 4% PEG8000 at 37 °C for 16 hours. After infection, the cells were washed with PBS and cultured for 10 days to measure the level of HBeAg or HBsAg.

Preparation of lentiviral vectors
293T cells were cotransfected with the transfer vector (1 μg), vesicular stomatitis virus G expression vector pCMV-VSV-G (0.25 μg), rev expression vector pRSV-Rev (0.25 μg), and gag-pol expression vector pCAG-HIVgp (0.75 μg) using TransIT-293 Transfection Reagent (TaKaRa Bio). After 24 hours, culture supernatants were harvested and filtered through a 0.45-μm filter.

Immunofluorescence analysis
Intracellular HBsAg was detected through immunofluorescence analysis. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and incubated with a mouse monoclonal anti-HBsAg antibody and Alexa Fluor 488-labeled secondary antibody targeting anti-mouse IgG (Thermo Fisher Scientific). Cells were observed under a fluorescence microscope (ECLIPSE Ts2; Nikon).

ELISA
HBsAg in the supernatant was determined using an ELISA kit (Beacle) according to the manufacturer’s instructions. HBeAg in the supernatant was determined using an ELISA kit (Abnova) according to the manufacturer’s instructions. A standard solution of HBeAg was purchased from Cell Biolabs.

Statistical analyses
Group comparisons were performed using the Student t test. Data are expressed as the mean±standard error of the mean. p values of <0.05 were considered statistically significant.

RESULTS

High-throughput screening of a compound library of 1827 FDA-approved drugs identified GSK3 inhibitors as anti-HBV drugs
We established a system to produce recombinant HBV-bearing NanoLuc gene in the genome to monitor the viral life cycle from entry to transcription.16 For this, we constructed 2 plasmids: (1) with 1.2-fold HBV genome encoding NanoLuc gene (p1.2xHBV-NL) and (2) carrying a replication-defective HBV genome (p1.2xHBV-Δepsilon).16 Upon transfection of these plasmids, HepG2 cells expressed pgRNA bearing the NanoLuc gene and HBV proteins, including HBc, HBs, pol, and HBx, from p1.2xHBV-NL and p1.2xHBV-Δepsilon, respectively. Recombinant HBV (HBV/NL) was secreted into the culture medium.
To establish a high-throughput system to identify inhibitors monitoring specific steps of the HBV life cycle from transcription to the release of infectious viral particles, we used a recombinant HBV reporter system with slight modifications.16 We constructed a plasmid, p1.2xHBV-secNL, to express pgRNA encoding the secreted form of NanoLuc (secNL) and a helper plasmid, p1.2xHBV-Δepsilon-d11, with an 11 amino acid deletion in the preS1 region of p1.2xHBV-Δepsilon, because this deletion increased the infectivity of HBV/NL.17

Using HepG2 cells transfected with these plasmids (illustrated in Supplemental Figure S1, http://links.lww.com/HEP/J694, Supplemental Method, http://links.lww.com/HEP/J727), we screened an FDA-approved drug library containing 1827 compounds to inhibit HBV production (Supplemental Figure S2, http://links.lww.com/HEP/J694). We identified 49 compounds with ≥5-fold reduction in HBV infectivity and ≥50% cell viability. These compounds included reverse transcriptase inhibitors (lamivudine, tenofovir, adefovir dipivoxil, and entecavir hydrate), telbivudine, and clevudine as well-known anti-HBV drugs (Figure 1A and Supplemental Table S1, http://links.lww.com/HEP/J694). Inhibition of Bcl-2 (ABT-199 and ABT-263) or CRM1 (KPT-330), which are known host factors,18,19 reduced HBV production. These results demonstrated that our system is functional and suitable for high-throughput screening. Notably, AZD1080, CHIR-98014, CHIR-98021, and BIO, which are known GSK3 inhibitors and uncharacterized as anti-HBV agents, suppressed HBV/secNL production without cytotoxic effects (Figures 1B and C). Therefore, we focused on GSK3 inhibitors for further experiments.

GSK3alpha/beta are host factors for HBV replication in HepAD38 cells
To assess whether the inhibition of GSK3 suppressed HBV production in HepAD38 cells, cells were cultured without tetracycline. At 6 days after incubation, the cells were treated with tetracycline for 24 hours to block the transcription of HBV pgRNA from integrated HBV DNA. The cells were treated with each compound in the presence of tetracycline for 5 days, and then HBeAg, HBsAg, and HBV RNA were quantified. These cells are supposed to transcribe pgRNA from HBV cccDNA but not from the integrated virus genome. Treatment with each GSK3 inhibitor (LY2090314 and CHIR-98014) reduced HBsAg, HBeAg, and HBV RNA (Figure 2A). The half-maximal effective concentration (EC50) of LY2090314 and CHIR-98014 for HBeAg was 0.48 and 1.87 μM, respectively (Supplemental Figure S3A, http://links.lww.com/HEP/J694). In addition, the EC50 value of LY2090314 and CHIR-98014 for HBsAg was 0.17 and 1.41 μM, respectively (Supplemental Figure S3B, http://links.lww.com/HEP/J694). The half-maximal cytotoxic concentration (CC50) of LY2090314 and CHIR-98014 in HepAD38 cells was 43.8 and 149.7 μM, respectively (Supplemental Figure S3C, http://links.lww.com/HEP/J694). Moreover, GSK3 inhibitors did not affect the level of cccDNA (Figure 2B), HBeAg (Supplemental Figure S3D, http://links.lww.com/HEP/J694), and intracellular core-associated HBV DNA (Supplemental Figure S3E, http://links.lww.com/HEP/J694) driven by the tetracycline-responsive promoter. These results suggest that GSK3 inhibitors suppress HBV transcription.
To determine which cellular targets (GSK3alpha and GSK3beta) of GSK3 inhibitors were involved in HBV transcription, we established GSK3alpha- and GSK3beta-deficient HepAD38 cell lines using the CRISPR-Cas9 system (Figure 2C). The deficiency of both GSK3alpha and GSK3beta significantly reduced the levels of HBsAg and HBeAg but did not affect the levels of cccDNA (Figure 2D). Importantly, exogenous expression of either GSK3alpha or GSK3beta in the GSK3alpha and GSK3beta double-knockout cells rescued the levels of both HBsAg and HBeAg in culture supernatants (Figure 2E). The deficiency of GSK3alpha and GSK3beta did not affect the level of HBeAg driven by the tetracycline-responsive promoter (Supplemental Figure S3F, http://links.lww.com/HEP/J694).

Inhibition of GSK3alpha/beta suppresses HBV transcription in HepG2-NTCP cells
To further examine the effect of GSK3 inhibitors on HBV replication, HepG2-NTCP cells were infected with HBV for 2 days. After infection, the cells were treated with 10 μM LY2090314 or 10 μM CHIR-98014 for 10 days. LY2090314 and CHIR-98014 significantly reduced the level of HBV antigens as monitored by measuring HBsAg and HBeAg in the culture medium (Figure 3A). In the cells, LY2090314 and CHIR-98014 treatment decreased HBsAg (Supplemental Figure S4, http://links.lww.com/HEP/J694). Northern blot analysis revealed that LY2090314 and CHIR-98014 significantly reduced the levels of the 2.4/2.1 and 3.5 kb HBV transcripts (Supplemental Figure S5A, http://links.lww.com/HEP/J694). To verify that GSK3alpha and GSK3beta influence HBV infection, we generated GSK3alpha and GSK3beta double-knockouts in HepG2-NTCP cells (Figure 3B). The levels of HBsAg and HBeAg, but not cccDNA, were lower in culture supernatants from GSK3alpha and GSK3beta double-knockout cells than those in culture supernatants from parental HepG2-NTCP cells (Figure 3C). The rescue of either GSK3alpha or GSK3beta in GSK3alpha and GSK3beta double-knockout cells restored the levels of both HBsAg and HBeAg. This effect was also observed by detecting HBV RNA (Supplemental Figure S5B, http://links.lww.com/HEP/J694). Moreover, GSK3alpha and GSK3beta were required for efficient HBeAg and HBsAg production, but not cccDNA formation, in Huh7-NTCP cells (Supplemental Figure S6, http://links.lww.com/HEP/J694). Thus, GSK3alpha and GSK3beta play important roles in HBV replication.

GSK3alpha/beta are host factors for HBV replication in PHHs
To confirm the contribution of GSK3alpha/beta in HBV replication in PHHs, shRNAs designed against GSK3alpha, GSK3beta, and control shRNA were transduced into PHHs using the lentiviral vector system. Transduction of shGSK3alpha and GSK3beta reduced GSK3alpha and GSK3beta expression (Supplemental Figure S7A, http://links.lww.com/HEP/J694). Consistent with the result in Figure 3, the knockdown of GSK3alpha and GSK3beta expression reduced HBsAg (Supplemental Figure S7B, http://links.lww.com/HEP/J694), HBeAg (Supplemental Figure S7C, http://links.lww.com/HEP/J694), and HBV DNA (Supplemental Figure S7D, http://links.lww.com/HEP/J694).
The effect of GSK3 inhibitors on HBV replication was investigated in PHHs, which were treated with several GSK3 inhibitors 48 hours after HBV infection. Ten days after treatment, HBV replication was evaluated by quantifying HBsAg and HBeAg in the culture supernatant (Figures 4A–F). All GSK3 inhibitors significantly reduced the levels of HBsAg and HBeAg. Notably, LY2090314 was highly potent against the reduction of both HBsAg and HBeAg, with EC50 values of 0.414 and 0.086 μM (Supplemental Table S2, http://links.lww.com/HEP/J694), respectively. In addition, the CC50 value of LY2090314 was 26.0 μM (Supplemental Figure S8, http://links.lww.com/HEP/J694). GSK3 inhibitors significantly decreased intracellular HBsAg (Supplemental Figure S9, http://links.lww.com/HEP/J694) and HBV RNA (Figures 4G and H). We next evaluated whether GSK3 inhibitors suppressed the level of extracellular HBV DNA. Entecavir strongly reduced the level of HBV DNA in the culture supernatant (Supplemental Figure S10A, http://links.lww.com/HEP/J694). Importantly, all GSK3 inhibitors significantly reduced the level of extracellular HBV DNA (Supplemental Figures S10B–G, http://links.lww.com/HEP/J694). Moreover, GSK3 inhibitors did not significantly reduce the level of cccDNA (Supplemental Figure S11, http://links.lww.com/HEP/J694). These results suggest that GSK3 inhibitors have potent suppressive activity against HBV transcription.

FOXK1 and FOXK2 are candidate regulators of HBV transcription
GSK3alpha and GSK3beta are Ser/Thr-specific kinases that phosphorylate several substrates, including Tau and beta-catenin.20 To elucidate how GSK3alpha and GSK3beta regulate HBV transcription, we performed phosphoproteome analysis using HepG2-NTCP cells, GSK3alpha/beta double-knockout HepG2-NTCP cells, and GSK3alpha/beta double-knockout HepG2-NTCP cells transduced with GSK3alpha or GSK3beta (Figure 5A and Supplemental Table S3, http://links.lww.com/HEP/J695). We searched for transcription factors phosphorylated by GSK3alpha or GSK3beta. We identified ATF2, TBX2, FOXK1, and FOXK2 as candidate regulators of HBV transcription (Figure 5B). To investigate the functional role of these proteins in HBV transcription, we used the CRISPR-Cas9 system to establish knockout HepG2-NTCP cells deficient in ATF2, TBX2, or FOXK1/2. ATF2 or TBX2 knockout cells exhibited no significant effect on the level of HBsAg and HBeAg (Figures 5C and D), suggesting that ATF2 and TBX2 are not involved in HBV transcription. By contrast, double knockout of both FOXK1 and FOXK2 reduced HBsAg and HBeAg more efficiently than that in parental HepG2-NTCP cells (Figure 5E). Therefore, we focused on FOXK1 and FOXK2 for further analysis.

Phosphorylation of FOXK2 is important for HBV transcription
To determine if FOXK1 or FOXK2 was involved in the regulation of HBV transcription, FOXK1 and FOXK2 double-knockout cells were transduced with EGFP, used as a control protein, FOXK1, FOXK2, or both FOXK1 and FOXK2 using a lentiviral vector (Figure 6A) and then infected with HBV. The levels of HBeAg and HBsAg were not restored by only FOXK1 expression (Figure 6B). However, FOXK2 expression rescued the production of HBeAg and HBsAg in FOXK1 and FOXK2 double-knockout cells, suggesting that FOXK2 positively regulates HBV transcription. To investigate whether the GSK3 inhibitor suppresses FOXK2 phosphorylation, HepG2-NTCP cells were treated with DMSO or LY2090314, and cell lysates were subjected to Phos-tag polyacrylamide gel electrophoresis, followed by immunoblot analysis. Treatment of LY2090314 reduced a higher molecular band when compared with the treatment of DMSO (Supplemental Figure S12, http://links.lww.com/HEP/J694). Because we identified that serine 424 in FOXK2 is phosphorylated by GSK3alpha and GSK3beta (Figure 5B), the roles of FOXK2-S424 phosphorylation on the production of HBeAg and HBsAg were analyzed by mutating S424 to alanine (S424A) or aspartic acid (S424D). FOXK2 mutations carrying S424A mimicked a constitutively dephosphorylated state, and S424D mimicked a constitutively phosphorylated state. Among them, no differences in the expression level of FOXK2 were observed in FOXK1 and FOXK2 double-knockout cells (Figure 6C). Consistently, the levels of HBeAg and HBsAg in FOXK1 and FOXK2 double-knockout cells expressing wild-type FOXK2 were comparable with those in HepG2-NTCP cells (Figure 6D). However, the expression of FOXK2 carrying an S424A mutation, FOXK2-S424A, in FOXK1 and FOXK2 double-knockout cells was impaired to restore the levels of HBeAg and HBsAg. Interestingly, the impairment of HBeAg and HBsAg production by FOXK1 and FOXK2 depletion was restored by the expression of FOXK2-S424D. To further investigate whether GSK3-mediated phosphorylation of FOXK2 is required for HBV transcription, GSK3alpha and GSK3beta double-knockout cells were transduced with FOXK2-WT or FOXK2-S424D and then infected with HBV. The expression of wild-type FOXK2 did not restore HBeAg and HBsAg in GSK3alpha and GSK3beta double-knockout cells (Figure 6E). However, the FOXK2-S424D mutant restored the production of HBV antigens. Importantly, the inhibitory effect of GSK3 inhibitor on the production of HBsAg and HBeAg was significantly impaired in FOXK1 and FOXK2 double-knockout cells expressing FOXK2-S424D compared with that in FOXK1 and FOXK2 double-knockout cells expressing wild-type FOXK2 (Figure 6F), suggesting that the GSK3-mediated phosphorylation of FOXK2-S424 plays an important role in the transcription of genes for HBe and HBs antigens.

FOXK2 binds HBV DNA
FOXK2 mainly binds a promoter region containing the 5′-GTAAACA-3′ sequence.21 Therefore, we searched for this motif in HBV genomic DNA using the HBV database. The 5′-GTAAACA-3′ sequence was at 1182–1188 bp within HBV enhancer I. A database of published HBV sequences showed that this motif is highly conserved across genotypes (>9000 sequences) (Supplemental Figure S13, http://links.lww.com/HEP/J694).22 To investigate whether FOXK2 binds to HBV DNA containing the 5′-GTAAACA-3′ sequence, we performed the gel mobility shift assay using extracts from FOXK1 and FOXK2 double-knockout cells that ectopically expressed HA-FOXK2. When the biotinylated HBV DNA fragment containing the 5′-GTAAACA-3′ sequence was used as a probe, we observed a shifted band in FOXK1 and FOXK2 double-knockout cells (Figure 7A, lane 2). This band was competed out by a cold competitor (Figure 7A, lane 3). Moreover, this band was enhanced by HA-FOXK2 expression (Figure 7A, compare lanes 2 and 6). The antibody against HA specifically supershifted this band (Figure 7A, compare lanes 4 and 8), suggesting that this complex contains FOXK2. To examine whether the FOXK2-HBV DNA complex is reduced by GSK3 inhibitors, we used HepG2-NTCP cell lysates treated with DMSO or LY2090314. The shifted band was fainter in LY2090314-treated cells than in DMSO-treated cells (Figure 7B). To further confirm the effect of GSK3 inhibitors on the binding of FOXK2 to HBV DNA, we performed a ChIP analysis. In HepG2-NTCP cells infected with HBV, FOXK2 bound to HBV DNA (Figure 7C). By contrast, the binding of FOXK2 to HBV DNA decreased in LY2090314-treated HepG2-NTCP cells infected with HBV. These results suggest that FOXK2 phosphorylation is important for the binding of FOXK2 to HBV DNA. To further investigate the molecular mechanism of a GSK3 inhibitor in HBV transcription regulation, we examined whether LY2090314 affects the binding of RNA polymerase II phosphorylated at Ser2 (pol II-pS2) or Ser5 (pol II-pS5), which are active transcription markers, to HBV DNA (Figures 7D and E). Treatment of LY2090314 reduced the level of pol II-pS2 and pol II-pS5 binding to HBV DNA. Moreover, FOXK2 binding is highly correlated with trimethylated histone H3 at Lys4 (H3K4me3) typically found at active promoters.23 Indeed, HBV DNA was shown to reduce H3K4me3 when cells were treated with LY2090314 (Figure 7F). These results suggested that GSK3 inhibitors can lead to transcriptional repression.

DISCUSSION
Assay systems, including ELISA, RT-qPCR, and blotting approaches, to assess HBV replication are unsuitable for high-throughput screening because of screening costs, technical limitations, and high background due to the contamination of input virus from the inoculum. To overcome these limitations, we developed recombinant HBV encoding NanoLuc to investigate the steps of HBV replication, including entry, internalization, disassembly, nuclear import, cccDNA biogenesis, and transcription from the core promoter of cccDNA.16 Cai et al24 and Verrier et al25 independently established HepDE19 and DHBV-HA2/3 cell lines, respectively, in which the production of haemagglutinin-tagged HBeAg was dependent on cccDNA. However, these approaches cannot identify host factors involved in the infectivity of HBV virion because of the low efficiency of HBV spread to neighboring cells.26

In this study, we established a novel reporter assay to identify anti-HBV drugs against the HBV life cycle from transcription to egress. Our approach exhibits a wide dynamic range because lamivudine dramatically reduced the level of HBV infectivity to 2.2% of that of DMSO-treated cells (Supplemental Table S1, http://links.lww.com/HEP/J694). Moreover, this system is highly sensitive because it is suitable for 96-well plates, allowing high-throughput screening to evaluate the late step of HBV replication. We applied this system to screen anti-HBV candidates from an FDA-approved drug library containing 1827 compounds and identified 49 compounds, including Bcl-2 inhibitors, which suppress HBV replication. Bcl-2 is required for HBV DNA replication at posttranscription steps. The binding of HBx to Bcl-2 is crucial for the increase in HBx-induced cytosolic calcium and HBV DNA replication.18 In our assay system, the Bcl-2 inhibitors ABT-199 and ABT-263 strongly suppressed HBV replication (Supplemental Table S1, http://links.lww.com/HEP/J694). The identification of KPT-330, a CRM1 inhibitor (Supplemental Table S1, http://links.lww.com/HEP/J694), which suppresses HBV pgRNA encapsidation or nuclear export of HBV RNA,19,27 confirmed the applicability of our system for the evaluation of HBV replication, including HBV pgRNA encapsidation, using a high-throughput approach.
We identified GSK3alpha/beta as host factors for HBV replication. GSK3alpha/beta has diverse functions, including regulation of differentiation, proliferation, and apoptosis as well as roles in Alzheimer’s disease (AD), diabetes, cancer, and inflammation.28 Moreover, GSK3 regulates toll-like receptor (TLR) signaling pathways. Inhibition of GSK3 prevents TLR-induced NF-κB activation, resulting in the suppression of cytokine production and apoptosis.29 HBV induces the stimulation of TLR2 signaling.30 TLR2 participates in the induction of inflammation during chronic HBV infection, leading to liver diseases, such as liver cirrhosis and HCC.31 TLR2 is associated with the development of hyperlipidemia,32 a risk factor for HCC. GSK3 is a known substrate of Tau. Hyperphosphorylation of Tau accumulates in neurofibrillary tangles, leading to neuronal cell death and AD pathology. Therefore, GSK3 is one of the promising therapeutic candidates for AD. Lithium salts, a direct inhibitor of GSK3, have been widely used for treating bipolar disorders in the past decades.33 Two clinical trials with lithium were conducted in participants with AD for up to 1 year without severe side effects, suggesting that the prescription of lithium for individuals is relatively safe.34 These results suggested that GSK3 inhibitors may be tolerated in long-term anti-HBV treatment. Thus, GSK3 is a potential therapeutic target for inhibiting HBV replication and HBV-induced inflammation in chronic hepatitis B.
GSK3alpha/beta targets >100 substrates and regulates not only various cellular processes, including metabolism, differentiation, and proliferation but also the viral replication cycle. GSK3 inhibitors suppress HCV virion maturation, release, and replication.35 GSK3 phosphorylates severe acute respiratory syndrome coronavirus N protein and supports viral replication.36 Inhibition of GSK3 phosphorylation levels by the AKT inhibitor prevents the entry of influenza virus.37 In this study, we showed that GSK3 plays an important role in HBV transcription by regulating FOXK2 phosphorylation. Notably, the anti-HBV activity of GSK3 inhibitors is not likely responsible for the effect of HBV DNA replication based on several observations. GSK3 inhibitors did not affect the level of cccDNA and intracellular core-associated HBV DNA in HepAD38 cells (Figures 2B and D; Supplemental Figure S3E, http://links.lww.com/HEP/J694). These results indicated that GSK3 does not regulate HBV DNA replication.
In this study, we showed that FOXK2 induces the production of HBeAg and HBsAg by binding to HBV enhancer I. This result is consistent with previous results that the HBV enhancer I element influenced the expression of both pC/pg and preS1/S2 transcripts.38,39 FOXK2 was initially identified as an interleukin-enhancer binding factor (ILF), which binds to 2 purine-rich domains within the HIV long terminal repeat,40 with preferential binding to GTAAACA. FOXK2 regulates various biological processes, including proliferation, differentiation, cell cycle progression, apoptosis, and cell metabolism.41 In human cancers, the expression of FOXK2 is low in glioma42 and breast cancer.43 By contrast, high FOXK2 expression in HCC is closely associated with larger tumors, advanced TNM stages, and tumor vascular invasion.44 miR-1271 targets the 3′UTR of FOXK2 mRNA to inhibit its expression in HCC. Overexpression of miR-1271 not only inhibited FOXK2 expression but also HBeAg, HBsAg, and HBV DNA production.45 These results suggest that FOXK2 is a potential tumor biomarker as well as a therapeutic target for HCC and HBV replication.
In conclusion, we show evidence for the roles of GSK3 and FOXK2 in HBV transcription. Our approach provides a powerful strategy for identifying host factors and pathways that regulate HBV replication during transcription, which can lead to the discovery of therapeutic targets for interrupting HBV replication in patients with chronic hepatitis B.

Supplementary Material