Tetracycline-inducible replications of wild-type and an adefovir-dipivoxil-resistant hepatitis B virus ;in human liver cells
10.3760/cma.j.issn.0253-9624.2016.04.013
- VernacularTitle:乙型肝炎病毒野生株和阿德福韦酯耐药变异株稳定表达细胞模型的建立和应用
- Author:
Jianghua WANG
1
;
Xueyan WANG
;
Ran FEI
;
Haiying ZHANG
;
Lai WEI
Author Information
1. 100044北京大学人民医院北京大学肝病研究所,丙型肝炎和肝病免疫治疗北京市重点实验室
- Keywords:
Hepatitis B virus;
Adefovir dipivoxil;
Tetracycline;
Mutation
- From:
Chinese Journal of Preventive Medicine
2016;50(4):351-356
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish cell lines with inducible replications of wild?type or rtE218G, an adefovir?dipivoxil?resistant HBV mutant. Methods Tetracycline transactivator (tTA) was stably transfected into human liver cell line HepG2.1.2 folds of full?length of wild?type or rtE218G?mutated HBV genomes were cloned into the pTRE vector and co?transfected into the tTA?expressing cells with a linear selection marker for hygromycin, respectively. After hygromycin screening, clones with the highest levels of tetracycline?inducible HBV replications were selected. The obtained cell lines were further used to evaluate the in vitro sensitivity of rtE218G mutant to adefovir?dipivoxil. Results HepG2?off23, a HepG2?derived cell line with stable tTA expression was established. PTRE?based plasmids carrying wild?type HBV (pTRE?HBV?WT) or rtE218G mutant (pTRE?HBV?E218GHBV) were constructed. After stable transfection of the HBV constructs into HepG2?off23 cells, cell lines with robust and tetracycline?inducible replications of wild?type HBV (HepG2?tetHBV?WT) and rtE218G?mutated HBV (HepG2?tetHBV?E218G) were selected. In the two cell lines, high levels of viral core protein and DNA replication could be detected after 144 hours of culture, which could be potently inhibited when tetracycline was added into the medium. At the presence of 1 000 ng/ml of tetracycline, HBV replication intermediates were hardly detected by Southern blotting experiments. HBV mutant with rtE218G could independently confer resistance to adefovir in vitro. IC50 for HBV rtE218G mutant of adefovir was (6.49±0.09)μmol/L, which was significantly higher than that for wild type virus (2.49 ± 0.05)μmol/L. Conclusion Wild?type and the rtE218G HBV mutant could be expressed and efficiently regulated by tetracycline in the established new cell lines. These cell lines could be useful tools for the HBV virology and anti?HBV drug screening studies.
