Cell lines

HepG2 (hepatoblastoma cell line, ATCC, HB-8065), Huh-7 (hepatocellular carcinoma cell line, Korea cell line bank, 60104), HepG2.2.15 (HBV-producing cells)37, HepG2-NTCP, and Huh-7-NTCP cells were cultured at 37 °C in 5% CO2 in Dulbecco′s Modified Eagle′s Medium with 10% FBS and 100 U penicillin with 0.1 mg per mL streptomycin. The same conditions were used to culture HepG2 and Huh-7 cells that were transfected with pHBV1.2× (genotype D). All maintained cells tested by PCR were mycoplasma-negative.

Quantitative PCR to measure intracellular HBV DNA and cccDNA

Cells were treated with the indicated compound(s), after which the HBV DNA that had been secreted into the medium, and intracellular HBV DNA and cccDNA, were extracted, purified and quantified by quantitative PCR. To isolate the secreted HBV DNA, culture media were collected and centrifuged at 15,000 × g to remove debris. The medium was diluted 1:1 with phosphate-buffered saline (PBS), and 1 M NaOH solution was added to a final concentration of 0.1 M. The mixture was incubated at 37 °C for 1 h. Protein was denatured by adding 2 M Tris-HCl (pH 7.5) solution to a final concentration of 0.2 M and incubating the mixture at 98 °C for 5 min. The protein precipitate was removed by centrifugation at 15,000 × g, and the supernatant was subjected to PCR with an HBV DNA primer and probe set (Supplementary Table 1). HBV DNA in the sera of mice infected with the replicative HBV plasmid pAAV-HBV1.2× (genotype C) was also measured by this method. To isolate intracellular HBV DNA, the cells were lysed on ice with lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% NP40) for 10 min. After removing cell debris and nuclei by centrifugation at 15,000 × g, the supernatant was treated with micrococcal nuclease (0.25 U per μL, M0247, New England Biolabs) at 37 °C for 1 h. The micrococcal nuclease was then inactivated by adding EGTA to a final concentration of 10 mM. Thereafter intracellular HBV DNA was extracted with a G-spinTM total kit (17046, Intron). The isolated intracellular HBV DNA was subjected to PCR with an HBV DNA primer and probe set (Supplementary Table 1). cccDNA was isolated by the Hirt extraction method54. Briefly, the cells were lysed in SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% SDS), and the lysates were mixed with KCl to a final concentration of 0.5 M. After incubation at 4 °C for 30 min, each lysate was centrifuged and the supernatant was extracted three times with phenol and once with chloroform. The DNA was then recovered by ethanol precipitation, and treated with T5 nuclease (10 U per μg for 0.5 h, M0363, New England Biolabs), and the enzyme was subsequently heat-denatured at 70 °C. The isolated cccDNA was subjected to PCR with a cccDNA primer and probe set (Supplementary Table 1). Quantification of cccDNA in liver tissue of HBV-infected PXB mice was also measured by this primer and probe set.

Southern blot analysis for detecting HBV DNA

HBV DNA was separated on a 1% agarose gel, blotted onto a nylon membrane (Hybond N + , RPN203B, GE Healthcare), and hybridized with a biotin-labeled full length HBV fragment prepared with the BioPrime™ DNA labeling system (18094011, ThermoFisher).

Infection of HepG2-NTCP and Huh-7-NTCP cells with HBV

HepG2-NTCP and Huh-7-NTCP cells were cultured in 12-well plates with the indicated compound(s) for 4 h, after which the cells were infected with HBV particles (107 viral genome equivalent per mL, genotype D) and treated with the indicated compound(s) for another 16 h. After removing the virus-containing medium, the cells were cultured in the presence of the indicated compound(s) for another 14 days.

Quantitative RT-PCR for measuring HBV transcription

Quantitative RT-PCR with primers that detect three HBV regions (R1, R2, and R3, Supplementary Table 1) was used to measure HBV transcription. This RT-PCR detects pgRNA and precore RNA (3.5 kB), two HBV surface-encoding RNAs (2.5 kB and 2.1 kB), and HBx-encoding RNA (0.7 kB)55. PCR conditions were 95 °C for 10 min initially, then 50 cycles of 95 °C for 30 s, 60 °C for 60 s and 72 °C for 30 s, and finally 1 cycle of 95 °C for 60 s, 55 °C for 30 s and 95 °C for 30 s.

Analysis of intracellular HBV capsid assembly

Huh-7 cells were seeded in six-well plates at a density of 5 × 105 cells per well and transfected with pHBV1.2×, pCDNA3-Core (genotype C), or pCDNA3-Core Y118F plasmid DNA. After 12 h, the cells were incubated with the indicated compound(s) for 36 h. They were then lysed on ice with lysis buffer (1% NP40) for 10 min, and cell debris and nuclei were removed by centrifugation at 15,000 × g. The supernatant was laid on a sucrose gradient composed of 1 mL of 40% sucrose (wt per vol) and 1.5 mL of 20% sucrose in 1 × PBS, and the HBV capsids were sedimented by centrifugation at 400,000 × g and 20 °C for 8 h. The pellets were resuspended in 50 μL of 1 × PBS and sonicated (1 s per stroke × 3 times), and samples were separated on 1% agarose gels. The gels were transferred onto nitrocellulose membranes by capillary transfer in 10 × SSC, and HBV core particles were detected by immunoblot analysis using anti-HBV core antibody (1:2000, B0586, Dako).

Expression and purification of HBV core protein

Cp149 (amino acids 1–149) is a truncated form of the HBV core protein. It was expressed in Escherichia coli and the expressed protein was purified56. The gene encoding Cp149-Y132A with a C-terminal thrombin cleavage site was synthesized and optimized for expression in bacterial cells (Gene Universal). The gene was cloned between the NdeI and BamHI sites in pET23a vector containing a hexahistidine tag. Recombinant Cp149-Y132A was expressed in E. coli BL21 (DE3) cells that were cultured in LB medium at 37 °C. When the culture reached OD600 of 0.7–0.8, expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside, and the culture was cooled to 16 °C. The cells were harvested after 16 h, flash-frozen in liquid nitrogen, and stored at −80 °C. To purify Cp149-Y132A, thawed cells were resuspended in lysis buffer (20 mM Tris-HCl pH 9.0, 200 mM NaCl, 10 mM imidazole, 10 ug per mL DNaseI, 1 mM phenylmethylsulfonyl fluoride) and sonicated. The supernatant was loaded onto Ni-NTA affinity resin and the protein was eluted with a gradient of 20 to 500 mM imidazole in lysis buffer. After removing the histidine tag with thrombin, the protein was further purified by HiTrap Q anion exchange chromatography (17115401, GE Healthcare).

Immunoblot analysis of HBV capsid assembly in vitro

To determine effects on Cp149 assembly in vitro, compounds were added to Cp149-containing suspensions. The final concentration of Cp149 was 1 mg per mL and the compounds were added at 0.1–10 μM. The suspensions were then mixed with reaction buffer (150 mM HEPES, pH 7.5, 15 mM NaCl) added at a ratio of 2:1. In vitro assembly was allowed to progress at 37 °C for 1 h and the HBV capsids were detected by immunoblot analysis using anti-HBV core antibody (1:2000, B0586, Dako). Uncropped and unprocessed scans of the most important blots are provided in the Source Data file.

Sucrose density gradient analysis of HBV capsid assembly in vitro

Cp149 structures formed by assembly in the presence and absence of inhibitory compounds were subjected to sucrose density gradient analysis. Samples (150 μL) were laid on sucrose density gradients composed of 800 μL of 50% (wt per vol), 800 μL of 40%, 800 μL of 30%, 800 μL of 20%, and 650 μL of 10% sucrose in 150 mM HEPES, pH 7.5. After centrifugation at 250,000 × g and 20 °C for 1.5 h, ten 400 μL fractions were collected from top to bottom, and each fraction was analyzed by 15% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue R-250 or subjected to immunoblot analysis with anti-HBV core antibody. The densities of the individual bands were analyzed by ImageJ software.

Electron microscopy of HBV capsid assembly in vitro

Cp149 was assembled in reaction buffer (150 mM HEPES, pH 7.5, 15 mM NaCl) in the presence and absence of ciclopirox. Five microliters of the solution containing the assembled HBV core particles was negatively stained by incubation on a carbon-coated grid for 1 min, followed by washing with water and staining with 2% uranyl acetate for 1 min. The grids were examined with a Tecnai G2 F30 S-TWIN transmission electron microscope.

Crystallization and data collection

To co-crystallize HBV core protein with ciclopirox, concentrated Cp149-Y132A protein (50 mg per mL) was incubated with 5 mM ciclopirox on ice for 30 min in a buffer consisting of 20 mM Tris HCl pH 9.0 and 200 mM NaCl. Crystals of HBV core protein and ciclopirox were grown by the sitting-drop vapor diffusion method at 22 °C with a reservoir solution containing 100 mM ammonium citrate pH 6.0–7.0, 2–14% (wt per vol) PEG5000MME, 2% hexylene glycol, and 10% isopropanol. For data collection, the protein crystals were flash-frozen in liquid nitrogen with 20% glycerol, added as a cryoprotectant. X-ray diffraction datasets were collected at beamline 7A of the Pohang Accelerator Laboratory (PAL). The diffraction data were indexed, integrated, and scaled with the HKL2000 program package (HKL Research Inc.).

Structure determination

The initial phases were calculated by molecular replacement using the program Phaser57. The previously published structure of the HBV core protein complexed with NVR-010–001-E2 (PDB code 5E0I) was used as search probe28. Atomic models were built by iterative modeling and refinement using the programs COOT58, REFMAC559, and PHENIX60. All molecular figures were prepared with PyMol (www.pymol.org). The crystallographic data and refinement statistics are summarized in Table 1.

Cell viability

HepG2.2.15 cells and HepG2 cells transfected with the pHBV1.2× plasmid were seeded on 96-well plates at a density of 2 × 104 cells per well. After treatment with the indicated compound(s), the medium was removed and the cells were incubated at 37 °C for 1 h in EZ-CYTOX solution (EZ-5000, DoGenBio). The absorbance at 450 nm was then measured.


An HBsAg ELISA kit (KA0286, Abnova) was used to quantify HBsAg protein. Briefly, culture media were incubated in individual wells of the capture plates for 2 h. After washing, horseradish peroxidase-labeled rabbit anti-HBV surface antibody was added, followed by incubation for an additional 2 h. The substrate solution was added for 15–60 min and absorbance at 450 nm was measured.

Mouse model

Human liver-chimeric uPA/SCID (PXB) mice were generated by PhoenixBio Co., Ltd. (Higashi-Hiroshima, Japan)61. At age 3 weeks, uPA/SCID mice underwent transplantation of human hepatocytes (105–106 cells per mouse) via the spleen. At 9 weeks after transplantation, the replacement index in all the mice exceeded 90%. At age 17 weeks, the mice were transferred to the laboratory animal research center of CHA University where they received a single intravenous injection of human HBV virions (5 × 107 copies per mouse, genotype D). After 6 weeks, persistent HBV infection of the human hepatocytes was established in the chimeric liver. At 22 weeks of age the mice began daily oral treatment with ciclopirox (5 mg per kg). TDF (5 mg per kg) was also administered orally every day. Serum samples were collected from the orbital sinus each week to measure HBsAg (WB-1296, Wantai Bio-Pharm), HBeAg (WB-2496, Wantai Bio-Pharm), HBV DNA, ALT (ab234578, Abcam), and human albumin (ab179887, Abcam). The animals were killed by CO2 gassing after 35 days. Liver tissue samples were analyzed to measure cccDNA, and sectioned for immunohistochemical staining with anti-HBV core antibodies (OAAI00128, Aviva Systems Biology). Intrahepatic cccDNA levels were determined in DNA extracted from liver specimens using a G-spin™ Total DNA extraction kit (17046, Intron) after digestion with T5 nuclease (10 U per μg for 0.5 h). For normalization, numbers of human hepatocytes in the liver specimens were estimated by measuring human hemoglobin beta (assay ID Hs00758889_s1, Thermo Fisher Scientific, USA). Animal procedures were performed in accordance with the guidelines of the Committee for Animal Experiments of CHA University. Infection, extraction of serum samples, and sacrifice were performed under ether anesthesia. All animal protocols were approved by the Institutional Animal Care and Use Committee of CHA University (IACUC-180090).

Statistical analysis

The bar-graph data show the means and standard deviations of one representative of 2–3 independent experiments. Groups were compared using two-tailed student’s t-test. P values of < 0.05 were considered significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.