Encyclopedia of Experiments: Immunology
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Transcript
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.
