This study investigates the infection mechanism of hepatitis B virus (HBV) in human liver cancer cells genetically modified to express the sodium taurocholate co-transporting polypeptide (NTCP). The research highlights the role of NTCP in facilitating HBV entry and the subsequent expression of viral proteins.
Take a human liver cancer cell culture, genetically modified to express sodium taurocholate co-transporting polypeptide or NTCP — a transmembrane receptor.
Add recombinant hepatitis B virus, or HBV. The virus comprises an outer envelope containing surface proteins and an inner nucleocapsid enclosing the genome. Insertion of a luciferase reporter gene inhibits the virus's reproductive ability.
Viral envelope protein binds to the host cell's NTCP receptor.
NTCP interacts with the epidermal growth factor receptor, EGFR, and oligomerizes, inducing EGFR-mediated endocytosis of the virus.
The viral envelope fuses with endosome membrane to release the nucleocapsid, which disassembles at the nuclear pore complex.
The viral genome enters the nucleus and converts to covalently closed circular DNA or cccDNA — a stable template — encoding viral proteins, including luciferase.
At predefined intervals, harvest the cells and lyse to release intracellular luciferase.
Introduce a reporter substrate. Luciferase oxidizes the substrate, producing a signal.
A post-infection increase in luciferase activity indicates continuous expression of the viral genome.
Plate HepG2 cells, stably expressing sodium taurocholate, co-transporting polypeptide, or NTCP that are susceptible to HBV infection, and incubate at 37 degrees Celsius and 5% CO2.
One day before infection, plate approximately 5 x 104 HepG2/NTCP cells into the wells of a 96-well collagen-coated plate in 0.1 milliliters of culture medium. Thaw recombinant HPV in a 37 degrees Celsius water bath, until there is a small bit of ice remaining in the vial.
Prepare medium for infection by combining 78 microliters of fresh culture medium, two microliters of DMSO, 10 microliters of 40% PEG8000 in 1X PBS, and 10 microliters of recombinant HPV. Add 100 microliters of recombinant HBV solution to each well of a 96-well plate.
One day after infection, use 300 microliters of PBS to wash the cell three times, to remove the contaminating reporter protein from the virus fraction. Add 200 microliters of culture medium containing 2% DMSO to the infected cells, and incubate for one week or less.
To carry out analysis of the HBV infection, one week after infection, use PBS to wash the infected cells three times. Add 50 microliters of lysis buffer to the infected cells. Rock the culture plate for five minutes, then, centrifuge at 2000 times g for five minutes. Add 50 microliters of the reporter substrate to a luminometer plate, then, add 20 microliters of the cell lysate to the plate, and mix by briefly shaking. Finally, place the plate in the luminometer and initiate reading.
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