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 (K_D) 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 (K_D) 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 alo....

Protocol

NOTE: The following procedures must be performed in a Class II biological hazard flow hood or a laminar-flow hood. The handling of HBV was ethically approved by the IRB (MURA2020/1545). See the Table of Materials for details about all solutions, reagents, equipment, and cell lines used in this protocol.

1. Preparing host cells (mature hepatocytes)

1. Culture hepatocytes (3.75 × 10⁵ cells HepaRG or imHC) and maintain in a 75 cm² culture flask with 10 mL of DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 U/mL penicillin, and 100 µg/m....

Results

Hepatic maturation features were observed, including binucleated cells and polygonal-shaped morphology (Figure 1), especially in the differentiated stage of imHC (Figure 1A). A large increase in NTCP expression was measured in d-HepaRG and d-imHC at 7-fold and 40-fold, respectively (Figure 1B). The highly glycosylated form of NTCP, postulated to confer susceptibility to HBV entry, was detected more in d-imHC than in d-HepaRG (

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 cell culture flask	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