An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection

Overview

This video demonstrates an in vitro technique to detect hepatitis B virus (HBV) infection via a recombinant HBV reporter system. Upon infecting host cells, the recombinant viral genome expresses the luciferase reporter enzyme, which produces bioluminescence upon oxidizing its substrate — helping detect viral infection.

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 or 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 ml of culture medium, they will be 70-90% confluent the next day.
2. Transfection
   1. Transfect HepG2 cells with 5 µg of pUC1.2HBV (hepatitis B virus) delta epsilon and 5 µg of pUC1.2HBV/NL15 using a transfection reagent as per manufacturer's instructions.
   2. The next day, remove the culture medium and add 10 ml of fresh culture medium.
   3. 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.
3. Purification of recombinant HBV
   1. Remove cell debris from the culture medium containing recombinant HBV by centrifugation (2,300 x g for 5 min).
   2. Pass the supernatant through a 0.45 µm membrane filter.
   3. 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 (ethylenediaminetetraacetic acid) pH 8.0 and 5 ml of 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.6 in 500 ml) to the culture medium containing recombinant HBV and mix gently. Incubate overnight at 4 °C.
   4. Centrifuge at 2,300 x g for 20 min at 4 °C.
   5. Discard the supernatant and dissolve the pellet in 0.5 ml of TNE (10 mM Tris (tris(hydroxymethyl)aminomethane), 50 mM NaCl, 1 mM EDTA).
   6. Remove the debris by centrifugation at 2,300 x g for 5 min.
   7. Load 0.5 ml of TNE containing recombinant HBV onto 0.8 ml of 20% sucrose in TNE.
   8. Centrifuge at 100,000 × g for 3 hr at 15 °C.
   9. 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.
   10. Incubate overnight at 4 °C.
   11. 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

1. Culture HepG2 cells stably expressing NTCP (sodium taurocholate co-transporting polypeptide) (HepG2/NTCP) 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 (geneticin) and 100 U/ml nonessential amino acids at 37 °C in a humidified 5% CO₂ incubator.
2. One day before infection, plate approximately 5 x 10⁴ HepG2/NTCP cells into a 96-well collagen-coated plate in 0.1 ml of culture medium.
3. Thaw recombinant HBV in a 37 °C water bath until there is a small bit of ice remaining in the vial.
4. 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.
5. Add 100 µl of recombinant HBV solution to one well of the 96-well plate.
6. 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.
7. Incubate infected cells in 200 µl of culture medium containing 2% DMSO for one week or less.

3. Analysis

1. One week after infection, wash the infected cells 3 times with PBS.
2. Add 50 µl of lysis buffer to the infected cells.
3. Rock the culture plate for 5 min, and then centrifuge at 2,000 x g for 5 min.
4. Add 50 µl of the reporter substrate into the luminometer plate.
5. Add 20 µl of cell lysate to a luminometer plate containing the reporter substrate. Mix by vortexing briefly.
6. Place the plate in the luminometer and initiate the reading.

Materials

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 5xBuffer	Promega	E1941
GloMax 96 Microplate Luminometer	Promega	E6501
Sucrose	Nacalai tesque	30403-55
Luminometer plate	Greiner bio-one	655075
HepG2-NTCP1-myc-clone22	-	-	Nishitsuji, H., et al. Cancer. Sci. (2015)
pUC1.2HBV delta epsilon	-	-	Nishitsuji, H., et al. Cancer. Sci. (2015)
pUC1.2HBV/NL	-	-	Nishitsuji, H., et al. Cancer. Sci. (2015)
50 ml tube	Violamo	1-3500-02
Anti-Myc antibody	Sigma-Aldrich	C3956
HBIG	Japan Blood Products Organization	-
IFN-β	Mochida Pharmaceutical	1.49872E+13
Heparin	Sigma-Aldrich	H3393