Materials and reagents

1. Production of HBVcc

   1. HBV molecular clone plasmid

      A replication-competent HBV molecular clone with a 1.38-fold genome length of HBV genotype C strain (accession number: AB246345) (Figure 1) (Murayama et al., 2021) inserted into the pUC19 plasmid is prepared. This clone is introduced with a precore stop mutation (G1896A) to evaluate the production of HBc proteins by measuring the HBcrAg level excluding HBeAg. HBeAg is not produced from this construct.

      
      

      
      Figure 1. Structure of the hepatitis B virus (HBV) molecular clone for cell culture-generated HBV (HBVcc). Structure of the plasmid of the HBV molecular clone. This plasmid contains a 1.38-fold HBV genome (4,438 bp) of the HBV genotype C strain. This construct generates pregenomic RNA and expresses all HBV proteins.

      
      

   2. Sodium taurocholate cotransporting polypeptide (NTCP)-transduced cells

      HepG2-NTCPsec+ (provided by Dr. Marc Peter Windisch; Institut Pasteur Korea, Seoul, South Korea) (König et al., 2019). These cells were cultured with HepG2-NTCPsec+ culture medium (see Recipes) supplemented with Blasticidin (5µ g/mL)

   3. Dulbecco’s modified Eagle medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 044-29765)

   4. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F7524)

   5. MEM non-essential amino acids solution (100×) (NEAA) (Thermo Fisher Scientific, Gibco, catalog number: 11140050)

   6. HEPES (1 M) (Thermo Fisher Scientific, Gibco, catalog number: 15630130)

   7. Sodium pyruvate (100 mM) (Thermo Fisher Scientific, Gibco, catalog number: 11360070)

   8. Penicillin-streptomycin (10,000 U/mL) (Thermo Fisher Scientific, Gibco, catalog number: 15140122)

   9. Blasticidin S hydrochloride, HEPES solution (Blasticidin) (10 mg/mL) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 022-18713)

   10. Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Gibco, catalog number: 31985070)

   11. Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Invitrogen, catalog number: L3000015)

   12. Collagen-coated T225 flask (Corning, catalog number: NCO431082)

   13. Dulbecco’s phosphate buffered saline (PBS) (Nacalai Tesque, catalog number: 14249-24)

   14. Dimethyl sulfoxide (DMSO) (Nacalai Tesque, catalog number: 09659-14)

   15. Syringe-top filter unit; Millex-HV Syringe Filter Unit, 0.45 μm, PVDF, 33 mm (Merck Millipore, catalog number: SLHVR33RS)

   16. Amicon Ultra-15 centrifugal filter units (100 kDa) (Merck Millipore, catalog number: UFC910096)




2. Purification of HBVcc

   1. Optiprep (60% iodixanol solution) (Serumwerk Bernburg, catalog number: AXS-1114542)

   2. Open-top thinwall ultra-clear tube (14 mm × 89 mm) (Beckman Coulter, catalog number: 344059)

   3. DNase; RQ1 RNase-free DNase (Promega, catalog number: M6101)

   4. QIAamp DNA Mini kit (Qiagen, catalog number: 51306)

   5. Lumipulse G HBsAg-Quant (Fujirebio, catalog number: 296851)

   6. Lumipulse G HBcrAg (Fujirebio, catalog number: 294109)

   7. Luna Universal qPCR Master Mix (New England Biolabs, catalog number: M3003)

   8. Primers and probe for the real-time PCR targeting the HBs region:

      Forward primer: 5′-CTTCATCCTGCTGCTATGCCT-3′

      Reverse primer: 5′-AAAGCCCAGGATGATGGGAT-3′

      Probe: 5′-FAM-ATGTTGCCCGTTTGTCCTCTAATTCCA-TAMRA-3′

   9. TNE buffer (see Recipes)




3. Titration of HBVcc

   1. Collagen-coated 96-well culture plate (Corning, catalog number: NCO3585)

   2. Polyethylene glycol average mol wt 8,000 (PEG8000) (Sigma-Aldrich, catalog number: P2139)

   3. 4% paraformaldehyde phosphate buffer solution (4% PFA) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 163-20145)

   4. Block ACE (Bio-Rad, catalog number: BUF029)

   5. Triton X-100 detergent (Calbiochem, Merck Millipore, catalog number: 648466)

   6. Anti-HBc antibody; anti-hepatitis B virus core antigen IgG fraction (polyclonal) (AUSTRAL Biologicals, catalog number: HBP-023-9)

   7. Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 555 (Thermo Fisher Scientific, catalog number: A327320

   8. DAPI (4’,6-Diamidino-2-phenylindole dihydrochloride) (FUJIFILM Wako Pure Chemical Corporation, catalog number: 342-07431)

   9. 4% PEG8000 (see Recipes)

Recipes

1. HepG2-NTCPsec+ culture medium

   DMEM

   10% FBS

   Penicillin-streptomycin (100 U/mL)

   NEAA (1×)

   HEPES (10 mM)

   Sodium pyruvate (1 mM)




2. TNE buffer

   150 mM NaCl

   10 mM Tris HCl (pH 7.5)

   1 mM EDTA




3. 4% PEG8000

   Dissolve in water to 4% (w/v) in a water bath at 60 °C.

Equipment

1. Refrigerated centrifuge with swing rotor (TOMY, model: AX-310)

2. Automated chemiluminescent enzyme immunoassay system (Fujirebio, model: LUMIPULSE G1200)

3. Ultracentrifuge apparatus, Optima L-90K with an SW-41 Ti rotor (Beckman Coulter, model: Optima L-90K)

4. Analytical balance (Sartorius Lab Instruments, model: SECURA124-1SJP)

5. Real-time PCR system (Thermo Fisher Scientific, model: StepOnePlus Real-Time PCR System)

6. Fluorescence microscope (KEYENCE, model: BZ-X710)

Procedure