Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Summary

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Abstract

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Introduction

Chronic infection with hepatitis B virus (HBV) is a major risk factor for chronic liver diseases¹. Although current therapeutic strategies are based on nucleotide analogs that inhibit HBV pol function and/or the administration of type I interferon that activates immune responses in infected individuals as well as indirectly suppressing HBV proliferation through interferon-stimulated gene functions², these treatments cannot eliminate HBV DNA completely³. Moreover, the emergence of HBVs that are resistant to anti-pol agents is of concern⁴. The administration of combined anti-....

Protocol

1. Production of Recombinant HBV Encoding the Reporter Protein

1. Preparation of HepG2 cells
   1. Prepare cell culture medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml nonessential amino acids).
   2. Plate 4 x 10⁶ HepG2 cells in a 10-cm collagen-coated dish in 10 ml of culture medium the day before transfection. Incubate HepG2 cells at 37 °C in a humidified 5% CO₂ incubator.
      NOTE: When approximately 4 x 10⁶ HepG2 cells are plated in a 10 cm collagen-coated dish in 10....

Results

Figure 1 shows a schematic of the HBV genome, transcribed RNAs, virus proteins and their coding region on the genome. The reporter gene and its location in the genome are also indicated. Figure 2 shows the HBV reporter plasmid and helper plasmid containing the reporter gene. The pUC1.2HBV/NL reporter was constructed by deleting nucleotide positions 223-811 from the transcription initiation site of the preCore mRNA of pUC1.2HBV¹⁶ an.......

Discussion

The HBV genome has four primary open reading frames (ORFs) including the core, polymerase, surface and X ORFs (Figure 1). Transcription of these four HBV ORFs is tightly regulated by four promoters: the precore/core promoter containing enhancer II, S1 promoter, S2 promoter and X promoter containing enhancer I¹⁸. The 3.5 kb and 3.4 kb mRNAs are translated into PreCore and Core proteins, respectively. The large envelop protein (L) is produced from the largest subgenomic mRNA (2.5 kb.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Nano-Glo Luciferase Assay Regent	Promega	N1110
Penicillin-Streptomycin Mixed Solution	Nacalai tesque	09367-34
MEM Non-Essential Amino Acids Solution	Thermo Fisher Scientific	11140050
DMEM	Thermo Fisher Scientific	11995065
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31985070
100 mm/collagen-coated dish	Iwaki	4020-010
Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000001
Polyethylene glycol (PEG) 6000	Sigma-Aldrich	81255
Polyethylene glycol (PEG) 8000	Sigma-Aldrich	89510
NaCl	Nacalai tesque	31319-45
0.5 mol/L EDTA Solution	Nacalai tesque	06894-14
Tris-HCl	Nacalai tesque	35434-21
Millex-HP, 0.45 μm, polyethethersulfone, filter	Merck Millipore	SLHP033RS
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650
Collagen coated 96-well plate	Corning	NO3585
Passive Lysis 5x Buffer	Promega	E1941
GloMax 96 Microplate Luminometer	Promega	E6501
Sucrose	Nacalai tesque	30403-55
Luminometer plate	Greiner bio-one	655075
HepG2-NTCP1-myc-clone22	-	-	Reference 15
pUC1.2HBV delta epsilon	-	-	Reference 15
pUC1.2HBV/NL	-	-	Reference 15
50 ml tube	Violamo	1-3500-02
Anti-Myc antibody	Sigma-Aldrich	C3956
HBIG	Japan Blood Products Organization	-
IFN-β	Mochida Pharmaceutical	14987224005413
Heparin	Sigma-Aldrich	H3393