Dianxing Sun1, Michael Nassal. 1. University Hospital Freiburg, Internal Medicine II/Molecular Biology, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
Abstract
BACKGROUND/AIMS: Hepatitis B virus (HBV) cannot be propagated in cultured cells but two human hepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations. METHODS: Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals. RESULTS: HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals. CONCLUSIONS: The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background.
BACKGROUND/AIMS: Hepatitis B virus (HBV) cannot be propagated in cultured cells but two humanhepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations. METHODS: Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals. RESULTS: HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals. CONCLUSIONS: The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background.
Authors: Alexander Hoh; Maximilian Heeg; Yi Ni; Anita Schuch; Benedikt Binder; Nadine Hennecke; Hubert E Blum; Michael Nassal; Ulrike Protzer; Maike Hofmann; Stephan Urban; Robert Thimme Journal: J Virol Date: 2015-05-13 Impact factor: 5.103
Authors: Konduru S R Sastry; Chien Tei Too; Kaval Kaur; Adam J Gehring; Lionel Low; Alia Javiad; Teresa Pollicino; Li Li; Patrick T F Kennedy; Uri Lopatin; Paul A Macary; Antonio Bertoletti Journal: J Virol Date: 2010-12-15 Impact factor: 5.103