A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target
Summary
We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).
Abstract
Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.
Introduction
Hepatitis B virus (HBV) infection is considered a life-threatening disease worldwide. Chronic HBV infection is laden with a risk of liver cirrhosis and hepatocellular carcinoma1. Current anti-HBV treatment focuses mostly on post viral entry using nucleos(t)ide analogs (NAs) and interferon-alpha (IFN-α)2,3. The discovery of an HBV entry inhibitor, Myrcludex B, has identified a novel target for anti-HBV agents4. The combination of entry inhibitors and NAs in chronic HBV has significantly lessened the viral load compared to those targeting viral replication alone5,6. However, the classical hepatocyte model for the screening of HBV entry inhibitors is limited by low viral receptor levels (sodium taurocholate cotransporting polypeptide, NTCP). The overexpression of hNTCP in hepatoma cells (i.e., HepG2 and Huh7) improves HBV infectivity7,8. Nevertheless, these cell lines express low levels of phase I and II drug-metabolizing enzymes and exhibit genetic instability9. Hepatocyte models that can help target distinct mechanisms of candidate anti-HBV compounds such as previral entry, NTCP binding, and viral entry would expedite the identification and development of efficacious combination regimens. The study for anti-HBV activity of curcumin has elucidated the inhibition of viral entry as a new mechanism in addition to post viral entry interruption. This protocol details a host model for the screening of anti-HBV entry molecules10.
The goal of this method is to explore candidate anti-HBV compounds for viral entry inhibition, especially blocking NTCP binding and transport. As NTCP expression is a critical factor for HBV entry and infection, we optimized the hepatocyte maturation protocol to maximize NTCP levels11. In addition, this protocol can differentiate the inhibitory effect on HBV entry as inhibition of HBV attachment versus inhibition of internalization. The taurocholic acid (TCA) uptake assay was also modified using an ELISA-based method instead of a radioisotope to represent NTCP transport12,13. The receptor and ligand interaction was confirmed by their 3D structures14,15. The inhibition of NTCP function can be evaluated by measuring TCA uptake activity16. However, this technique did not provide direct evidence of NTCP binding to the candidate inhibitors. Therefore, the binding can be investigated using various techniques, such as surface plasmon resonance17, ELISA, fluorescence-based thermal shift assay (FTSA)18, FRET19, AlphaScreen, and various other methods20. Among these techniques, ITC is a goal standard in binding analysis because it can observe heat absorption or emission in almost every reaction21. The binding affinity (KD) of NTCP and candidate compounds was directly evaluated using ITC; these affinity values were more precise than those obtained using the in silico prediction model22.
This protocol covers techniques in hepatocyte maturation, HBV infection, and screening for HBV entry inhibitor. Briefly, a hepatocyte model was developed based on imHC and HepaRG cell lines. The cultured cells were differentiated into mature hepatocytes within 2 weeks. The upregulation of NTCP levels was detected using real-time PCR, western blot, and flow cytometry11. Hepatitis B virion (HBVcc) was produced and collected from HepG2.2.15. The differentiated imHC or HepaRG (d-imHC, d-HepaRG) was prophylactically treated with the anti-HBV candidates 2 h prior to the inoculation with HBV virion. The expected outcome of the experiment was the identification of the agents that decrease cellular HBV and infectivity. Anti-NTCP activity was evaluated using the TCA uptake assay. NTCP activity could be suppressed by the agents that specifically bound NTCP. The ITC technique was employed to investigate the feasibility of interactive binding that could predict inhibitors and their target proteins, determining the binding affinity (KD) of the ligand for the receptor via non-covalent interactions of the biomolecular complex23,24. For instance, KD ≥ 1 × 103 mM represents weak binding, KD ≥ 1 × 106 µM represents moderate binding, and KD ≤ 1 × 109 nM represents strong binding. The ΔG is directly correlated with binding interactions. In particular, a reaction with negative ΔG is an exergonic reaction, indicating that binding is a spontaneous process. A reaction with a negative ΔH indicates that the binding processes depend on hydrogen bonding and Van der Waals forces. Both TCA uptake and ITC data could be used to screen for anti-HBV entry agents. The outcomes of these protocols can provide a foundation for not only anti-HBV screening but also the interaction with NTCP as assessed through binding affinity and transport function. This paper describes host cell preparation and characterization, experimental design, and evaluation of the anti-HBV entry together with the NTCP binding affinity.
Protocol
Representative Results
Discussion
HBV infection is initiated via low-affinity binding to heparan sulfate proteoglycans (HSPGs) on hepatocytes25, followed by the binding to NTCP with subsequent internalization through endocytosis26. As NTCP is a crucial receptor for HBV entry, targeting HBV entry can be clinically translated to diminish de novo infection, mother-to-child transmission (MTCT), and recurrence after liver transplantation. Interrupting viral entry would be a feasible alternative cure for…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research project is supported by Mahidol University and the Thailand Science Research and Innovation (TSRI) separately awarded to A. Wongkajornsilp and K. Sa-ngiamsuntorn. This work was financially supported by the Office of National Higher Education Science Research and Innovation Policy Council through the Program Management Unit for Competitiveness (grant number C10F630093). A. Wongkajornsilp is a recipient of a Chalermprakiat grant of the Faculty of Medicine Siriraj Hospital, Mahidol University. The authors would like to thank Miss Sawinee Seemakhan (Excellent Center for Drug Discovery, Faculty of Science, Mahidol University) for her assistance with the ITC technique.
Materials
| Cell lines | |||
| HepaRG Cells, Cryopreserved | Thermo Fisher Scientific | HPRGC10 | |
| Hep-G2/2.2.15 Human Hepatoblastoma Cell Line | Merck | SCC249 | |
| Reagents | |||
| 4% Paraformadehyde Phosphate Buffer Solution | FUJIFLIM Wako chemical | 163-20145 | |
| BD Perm/Wash buffer | BD Biosciences | 554723 | Perm/Wash buffer |
| Cyclosporin A | abcam | 59865-13-3 | |
| EDTA | Invitrogen | 15575-038 | 8 mM |
| G 418 disulfate salt | Merck | 108321-42-2 | |
| Halt Protease Inhibitor Cocktail EDTA-free (100x) | Thermo Scientific | 78425 | |
| HEPES | Merck | 7365-45-9 | |
| illustraTM RNAspin Mini RNA isolation kits | GE Healthcare | 25-0500-71 | |
| illustra RNAspin Mini RNA Isolation Kit | GE Healthcare | 25-0500-71 | |
| ImProm-II Reverse Transcription System | Promega | A3800 | |
| KAPA SYBR FAST qPCR Kit | Kapa Biosystems | KK4600 | |
| Lenti-X Concentrator | Takara bio | PT4421-2 | concentrator |
| Luminata crescendo Western HRP substrate | Merck | WBLUR0100 | |
| Master Mix (2x) Universal | Kapa Biosystems | KK4600 | |
| Nucleospin DNA extraction kit | macherey-nagel | 1806/003 | |
| Phosphate buffered saline | Merck | P3813 | |
| Polyethylene glycol 8000 | Merck | 25322-68-3 | |
| ProLong Gold Antifade Mountant | Thermo scientific | P36930 | |
| Recombinant NTCP | Cloud-Clone | RPE421Hu02 | |
| RIPA Lysis Buffer (10x) | Merck | 20-188 | |
| TCA | Sigma | 345909-26-4 | |
| TCA Elisa kit | Mybiosource | MB2033685 | |
| Triton X-100 | Merck | 9036-19-5 | |
| Trypsin-EDTA | Gibco | 25200072 | Dilute to 0.125% |
| Antibodies | |||
| Anti-NTCP1 antibody | Abcam | ab131084 | 1:100 dilution |
| Anti-GAPDH antibody | Thermo Fisher Scientific | AM4300 | 1:200,000 dilution |
| HRP-conjugated goat anti-rabbit antibody | Abcam | ab205718 | 1:10,000 dilution |
| HRP goat anti-mouse secondary antibody | Abcam | ab97023 | 1:10,000 dilution |
| Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 | Invitrogen | A-11008 | 1:500 dilution |
| Reagent composition | |||
| 1° Antibody dilution buffer | |||
| 1x TBST | |||
| 3% BSA | Sigma | A7906-100G | Working concentration: 3% |
| Sodium azide | Sigma | 199931 | Working concentration: 0.05% |
| Hepatocyte Growth Medium | |||
| DME/F12 | Gibco | 12400-024 | |
| 10% FBS | Sigma Aldrich | F7524 | |
| 1% Pen/Strep | HyClon | SV30010 | |
| 1% GlutaMAX | Gibco | 35050-061 | |
| Hepatic maturation medium | |||
| Williams’ E medium | Sigma Aldrich | W4125-1L | |
| 10% FBS | Sigma Aldrich | F7524 | |
| 1% Pen/Strep | HyClon | SV30010 | |
| 1% GlutaMAX | Gibco | 35050-061 | |
| 5 µg/mL Insulin | Sigma Aldrich | 91077C-100MG | |
| 50 µM hydrocotisone | Sigma Aldrich | H0888-1g | |
| 2% DMSO | PanReac AppliChem | A3672-250ml | |
| IF Blocking solution | |||
| 1x PBS | Gibco | 21300-058 | |
| 3% BSA | Sigma | A7906-100G | Working concentration: 3% |
| 0.2% Triton X-100 | Sigma | T8787 | Working concentration: 0.2% |
| RIPA Lysis Buffer Solution | Merck | 20-188 | Final concentration: 1X |
| Protease Inhibitor Cocktail | Thermo Scientific | 78425 | Final concentration: 1X |
| Na3VO4 | Final concentration: 1 mM | ||
| PMSF | Final concentration: 1 mM | ||
| NaF | Final concentration: 10 mM | ||
| Western blot reagent | |||
| 10x Tris-buffered saline (TBS) | Bio-Rad | 170-6435 | Final concentration: 1X |
| Tween 20 | Merck | 9005-64-5 | |
| 1x TBST | 0.1% Tween 20 | ||
| 1x PBS | Gibco | 21300-058 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | A53225 | |
| Polyacrylamide gel | Bio-Rad | 161-0183 | |
| Ammonium Persulfate (APS) | Bio-Rad | 161-0700 | Final concentration: 0.05% |
| TEMED | Bio-Rad | 161-0800 | Stacker gel: 0.1%, Resolver gel: 0.05% |
| 2x Laemmli Sample Buffer | Bio-Rad | 161-0737 | Final concentration: 1X |
| Precision Plus Protein Dual Color Standards | Bio-Rad | 161-0374 | |
| WB Blocking solution/ 2° Antibody dilution buffer | |||
| 1x TBST | |||
| 5% Skim milk (nonfat dry milk) | Bio-Rad | 170-6404 | Working concentration: 5% |
| 1x Running buffer 1 L | |||
| 10x Tris-buffered saline (TBS) | Bio-Rad | 170-6435 | Final concentration: 1X |
| Glycine | Sigma | G8898 | 14.4 g |
| SDS | Merck | 7910 | Working concentration: 0.1% |
| Blot transfer buffer 500 mL | |||
| 10x Tris-buffered saline (TBS) | Bio-Rad | 170-6435 | Final concentration: 1X |
| Glycine | Sigma | G8898 | 7.2 g |
| Methanol | Merck | 106009 | 100 mL |
| Mild stripping solution 1 L | Adjust pH to 2.2 | ||
| Glycine | Sigma | G8898 | 15 g |
| SDS | Merck | 7910 | 1 g |
| Tween 20 | Merck | 9005-64-5 | 10 mL |
| Equipments | |||
| 15 mL centrifuge tube | Corning | 430052 | |
| 50 mL centrifuge tube | Corning | 430291 | |
| Airstream Class II | Esco | 2010621 | Biological safety cabinet |
| CelCulture CO2 Incubator | Esco | 2170002 | Humidified tissue culture incubator |
| CFX96 Touch Real-Time PCR Detector | Bio-Rad | 1855196 | |
| FACSVerse Flow Cytometer | BD Biosciences | 651154 | |
| Graduated pipettes (10 mL) | Jet Biofil | GSP010010 | |
| Graduated pipettes (5 mL) | Jet Biofil | GSP010005 | |
| MicroCal PEAQ-ITC | Malvern | Isothermal titration calorimeters | |
| Mini PROTEAN Tetra Cell | Bio-Rad | 1658004 | Electrophoresis chamber |
| Mini Trans-blot absorbent filter paper | Bio-Rad | 1703932 | |
| Omega Lum G Imaging System | Aplegen | 8418-10-0005 | |
| Pipette controller | Eppendorf | 4430000.018 | Easypet 3 |
| PowerPac HC | Bio-Rad | 1645052 | Power supply |
| PVDF membrane | Merck | IPVH00010 | |
| T-75 A91:D106flask | Corning | 431464U | |
| Trans-Blot SD Semi-Dry Transfer Cell | Bio-Rad | 1703940 | Semi-dry transfer cell |
| Ultrasonic processor (Vibra-Cell VCX 130) | Sonics & Materials | ||
| Versati Tabletop Refrigerated Centrifuge | Esco | T1000R | Centrifuge with swinging bucket rotar |
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