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Podsumowanie

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-viral agents directly targeting the different steps of human immunodeficiency virus (HIV) or hepatitis C virus (HCV) life cycle was shown to successfully suppress or eradicate the virus(es). Similar to this idea, the development of anti-HBV agent(s) that act directly on the different stages of the HBV life cycle is important for establishing future HBV therapies.

In general, the establishment of a simple in vitro culture system of the target virus facilitates the development of anti-virus agents. However, there are at least two barriers to the development of in vitro culture systems to screen anti-HBV agents. The first is the lack of a convenient in vitro cell culture system for HBV infection/proliferation. Unlike other viruses, such as HIV and HCV, which are propagated in established cell lines, it is difficult to cultivate HBV in vitro because of experimental limitations including a narrow host range. The use of specific cell culture systems such as the human hepatoma cell line HepaRG, which is susceptible to HBV infection,^5,6,7 have been developed to overcome these problems. Moreover, PXB cells, isolated from urokinase-type plasminogen activator transgenic/SCID mice inoculated with primary human hepatocyte (PHH), were shown to be susceptible to HBV infection and replication⁸. However, HBV replication levels in HepaRG are dependent on the cellular differentiation state after culture, which can cause inconsistent and irreproducible results of HBV infection/replication levels. PXB is commonly used for HBV infection experiments but is limited by its availability. A tetracycline inducible HBV expression cell line, HepAD38, has also been widely used to study HBV replication, but this system only allows evaluation after transcription and not at the entry step of HBV infection⁹. Recently, the identification of sodium taurocholate cotransporting polypeptide (NTCP) as a functional receptor for HBV has allowed the development of a variable HBV culture system¹⁰. Indeed, NTCP expression in non-susceptible hepatocarcinoma cells such as Huh7 and HepG2 enables HBV infection¹⁰ and thus, the choice of HBV susceptible cell lines has been expanded, resolving many of the experimental limitations. The second problem is the lack of a simple assay system to evaluate HBV infection and replication. Evaluation of HBV infection is usually conducted by analyzing HBV DNA, RNA and proteins. However, quantification of these virus markers is time consuming, often costly and not always simple. Therefore, the development of a simple assay system, such as using a reporter gene, might overcome problems associated with HBV assay systems.

However, because the genome size that can be packaged into an HBV capsid is limited — less than 3.7 kb¹¹ — the size of a reporter gene should be as short as possible. Furthermore, the presence of multiple cis elements scattered throughout the genome, which are essential for viral replication, limits the positions available for insertion of the reporter gene into the genome. Several reports have attempted to insert foreign genes, including HIV-1 Tat, green fluorescent protein, and DsRed, into the HBV genome^11,12,13. However, these recombinant HBVs are not useful for screening HBV infection/replication, or for the high-throughput screening of factors affecting HBV infection/replication. This is mainly because of the low productivity of recombinant viruses and the reduced intensity of reporter gene expression caused by inefficient virus production.

To overcome these issues, we constructed a reporter HBV with a high yield of virus production. This virus is highly sensitive for monitoring the early stages of the HBV replication cycle, from entry to transcription. To achieve this, NanoLuc (NL) was chosen as a marker gene because it is a small (171 amino acids) engineered luminescent reporter¹⁴. Moreover, NL is approximately 150-fold brighter than firefly or Renilla luciferase, and the luminescent reaction is ATP-independent, suggesting that the false hit rate will be low for high-throughput screening. The production efficiency of the recombinant HBV is approximately 1/5 of the parent HBV, and similar to levels reported for previous HBV recombinant viruses; however, the brightness of NL overcomes virus productivity issues so it can be used for the mass screening of anti-HBV agents.

Screening of anti-HBV agents using primary hepatocytes, HepaRG, HepAD38 and NTCP-transduced hepatocytes might be useful for the screening of anti-HBV agents by conventional method(s). However, the system described here has various advantages such as simple handling, high sensitivity, and low cost for screening. These advantages are suitable for high throughput assays to develop and identify new HBV agents for therapeutic purposes.

Protokół

1. Production of Recombinant HBV Encoding the Reporter Protein Preparation of HepG2 cells 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). 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 ml of culture medium, they will be 70-90% confluent the next day. Transfection Transfect HepG2 cells with 5 µg of pUC1.2HBV delta epsilon15 and 5 µg of pUC1.2HBV/NL15 using a transfection reagent as per manufacturer's instructions. The next day, remove the culture medium and add 10 ml of fresh culture medium. One week after transfection, transfer the culture medium containing the recombinant HBV to a 50 ml-tube and proceed to step 1.3.1. Add 10 ml of fresh culture medium to the plate containing the transfected cells. NOTE: Production of the recombinant HBV is maintained for 4 weeks. The culture medium containing recombinant HBV can be stored at 4 °C for 1 month. Purification of recombinant HBV Remove cell debris from the culture medium containing recombinant HBV by centrifugation (2,300 x g for 5 min). Pass the supernatant through a 0.45 µm membrane filter. Add an equal volume of 26% PEG/1.5 M NaCl (polyethylene glycol 6000: 130 g, NaCl, 49 g, 1 ml of 0.5 M EDTA pH 8.0 and 5 ml of 1 M HEPES pH 7.6 in 500 ml) to the culture medium containing recombinant HBV and mix gently. Incubate overnight at 4 °C. Centrifuge at 2,300 x g for 20 min at 4 °C. Discard the supernatant and dissolve the pellet in 0.5 ml of TNE (10 mM Tris, 50 mM NaCl, 1 mM EDTA). Remove the debris by centrifugation at 2,300 x g for 5 min. Load 0.5 ml of TNE containing recombinant HBV onto 0.8 ml of 20% sucrose in TNE. Centrifuge at 100,000 × g for 3 hr at 15 °C. Discard as much of the supernatant as possible, and save the pellet. Resuspend it in 1 ml of serum free DMEM per 40 ml of starting culture medium. Incubate overnight at 4 °C. Filter through a 0.45 µm filter. Prepare 0.5 ml aliquots and store at -80 °C. NOTE: If reporter protein contamination of the original virus sample is observed, purify the virus by density gradient ultra-centrifugation of 5-30% sucrose in TNE at 100,000 x g for 2 hr, or CsCl density equilibrated centrifugation from 1.1-1.6 g/ml at 150,000 x g for 50 hr. 2. Infection of Recombinant HBV Culture HepG2 cells stably expressing NTCP (HepG2/NTCP)15 that are susceptible to HBV infection in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml G-418 and 100 U/ml nonessential amino acids at 37 °C in a humidified 5% CO2 incubator. One day before infection, plate approximately 5 x 104 HepG2/NTCP cells14 into a 96-well collagen coated plate in 0.1 ml of culture medium. Thaw recombinant HBV in a 37 °C water bath until there is a small bit of ice remaining in the vial. Prepare the medium for infection by combining the following:10 µl of 40% PEG8000 in 1x phosphate buffered saline (PBS), 2 µl of dimethyl sulfoxide (DMSO), 10 µl of recombinant HBV and 78 µl of fresh culture medium per well of a 96-well plate. Add 100 µl of recombinant HBV solution to one well of the 96-well plate. One day after infection, wash the infected cells 3 times with 300 µl PBS per well to remove the contaminating reporter protein from the virus fraction. Incubate infected cells in 200 µl of culture medium containing 2% DMSO for one week or less. 3. Analysis One week after infection, wash the infected cells 3 times with PBS. Add 50 µl of lysis buffer to the infected cells. Rock the culture plate for 5 min, and then centrifuge at 2,000 x g for 5 min. Add 50 µl of the reporter substrate into the luminometer plate. Add 20 µl of cell lysate to a luminometer plate containing the reporter substrate. Mix by vortexing briefly. Place the plate in the luminometer and initiate reading15.

Representative 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

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 5xBuffer	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