Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication

Abstract

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

A Slow Maturation Process Renders Hepatitis B Virus Infectious

“…Virus Production Viral particles were produced in the cell line HepAd38 harboring an over-length HBV genome under control of a tetracycline-repressed promoter element (tet-off system) (Ladner et al, 1997). Cells were propagated in DMEM-F12 supplemented with 10% fetal calf serum (FCS), penicillin (50 IU/ml), streptomycin (50 mg/ml), G418 (400 mg/ml), insulin (5 mg/ml), hydrocortisone (50 mM), and tetracycline (0.3 mg/ml) and seeded on 5-STACK cell factories (Corning; 3,180 cm 2 growth area).…”

“…Phenotypes of HBV Viral particles were produced in HepAd38 cells, a cell line inducibly expressing the HBV genome under control of a tetracycline-responsive element (Ladner et al, 1997). Culture supernatants were harvested after 3-4 days and subjected to automated heparin affinity chromatography (HAC; see Figure S1).…”

Functional dissection of an IFN-α/β receptor 1 promoter variant that confers higher risk to chronic hepatitis B virus infection

“…To investigate whether HBV have any effect on PARP-1 and/or IFNAR1 expression, we used RT-PCR and QRT-PCR to analyze PARP-1 and IFNAR1 transcript levels in HepG2 and HepAD38 cell cultures. HepAD38 is an inducible HBV-expressing cell line, which was stably transfected with pregenomic RNA of HBV subtype ayw into parental hepatoblastoma cell line HepG2 [19]. Therefore, the only difference between these two cells is the presence or absence of HBV.…”

Short hairpin RNA induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells

“…HepAD38 cells which produce HBV under the control of tetracycline-responsive CMV-IE promoter were a generous gift from professor C. Seeger (Fox Chase Cancer Center, PA) [14]. 293FT cells were purchased from Invitrogen (Carlsbad, CA).…”

“…HepAD38 cells were maintained as described [14]. On the day of transduction, cells were plated at a density of 5 Â 10 5 cells in 60-mm cell culture dishes and 1 Â 10 9 particles of lentivirus were added to the medium along with polybrene at final concentration of 4 lg/ml.…”