Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus☆
Introduction
Chronic infection with hepatitis B virus (HBV) is a leading cause of severe liver disease such as cirrhosis and hepatocellular carcinoma [1]. HBV is a small, enveloped DNA-containing virus that replicates through reverse transcription (reviewed in [2], [3], [4]). The 3.2 kb relaxed circular DNA (RC-DNA) genome in virions is converted in the host cell nucleus into covalently closed circular DNA (cccDNA), the template for the transcription of several subgenomic and genomic RNAs. The pregenomic RNA (pgRNA) serves as mRNA for the core protein (HBcAg) plus the viral reverse transcriptase; second, it is packaged into new capsids and converted into RC-DNA. The longer precore mRNA includes the precore start codon, giving rise to secreted 17 kDa hepatitis B e antigen (HBeAg). Progeny nucleocapsids may redeliver their genomes to the nucleus for cccDNA amplification, or interact with the surface proteins and be released into the circulation.
Current treatments, comprising type-I interferons (IFNs) or the nucleos(t)ide analogs lamivudine and adefovir, are of limited efficacy [5], [6], may have adverse side-effects [7], and select for drug-resistant virus variants [8], [9], [10], [11]. Faster progress in developing improved antivirals is hampered, inter alia, by the lack of practicable infection systems. Except for chimpanzees, experimental HBV infection is mainly restricted to primary hepatocytes from humans; recent, more feasible alternatives are primary tupaia hepatocytes (PTH) which are infectable by serum-derived HBV [12], [13], [14], [15] and Woolly Monkey HBV (WMHBV; [12], [16]), and the HepaRG cell line [17]; however, no net amplification of the virus is achieved. Hence transient transfection of HepG2 and/or Huh7 cells, two distinct, well-differentiated human hepatoma cell lines, with appropriate HBV vectors remains the most robust system to analyze HBV replication and its inhibition. However, particularly for applications requiring standardized conditions such as drug screening, stably HBV producing cells provide distinct advantages: essentially all cells are productive, inter-experiment variation is minimized, and identical cells producing identical virus are relatively easily maintained and expanded. One such cell line, HepG2.2.15 [18], is widely used in antiviral research. Derived from HepG2 cells by transfection of a complex four-copy HBV construct, it produces modest levels [19] of virus. More expedient would be stable cell lines in which HBV expression is regulatable, preferentially up to higher levels.
The TetOFF and TetON systems [20] exploit the tetracycline (Tet) dependent binding of the Tet repressor to its cognate tet operator DNA motifs (or Tet response elements, TREs). Fusion with an activation domain creates a Tet-responsive trans-activator (tTA) which specifically induces transcription from promoters that have TREs fused to a minimal promoter with low basal activity. For TetOFF, activation occurs in the absence of Tet. Upon addition of Tet, or its homolog doxycycline (Dox), transcription ceases (Fig. 1A).
Here we report the establishment and characterization of stable TetOFF hepatoma cell lines for the controlled production of HBV. Though the Tet-system has previously been used (e.g. [21]), our approach differs by two aspects: first, it included both HepG2 and Huh7 cells, providing access to two distinct cellular backgrounds. Second, rather than using cotransfection we settled for a two-step procedure by first generating stable tTA-expressing lines; this allowed to preselect the best-performing tTA clones for the subsequent introduction of a TRE-controlled HBV expression vector (Fig. 1A). Below we describe the generation and characteristics of several stably and inducibly HBV producing hepatoma lines obtained by this approach.
Section snippets
Plasmids
pTetOFF (originally reported as pUHD15-neo [22]) and the improved VP16 minimal domain vectors ptTA2, ptTA3, and ptTA4 [23], pTRE2-Hyg, pTRE-luc, and pSEAP2 were obtained from Clontech. The HBV expression vector pTRE-HBVT contains a 1.05× HBV genome (subtype ayw; [24]), as in plasmid pCH-9/3091 [25], except that the non-transcribed HBV DNA from nucleotide (nt) 3091 to 3099 (numbering based on the core start [26]) was replaced by a 2 nt shorter linker (Fig. 1A); this removed the precore
Establishment of tTA expressing Huh7 and HepG2 cells
The Huh7 tTA clones H7TA61 and H7TA46.2 used here had been generated by transfection with the original pTetOFF plasmid. Corresponding HepG2 lines were not obtained, as also seen by others [32], but the modified tTAs (tTA 2, 3 and 4) [23] yielded numerous G418-resistant colonies. Clones providing high level and tightly regulated expression were identified via transient cotransfection with a Tet-controlled luciferase plus a Tet-independent secreted alkaline phosphatase (SEAP) expression plasmid.
Discussion
Despite only modest and not easily controlled levels of HBV expression, HepG2.2.15 [18] is still the single most widely used HBV producing cell line in antiviral research. The new cell lines described here extend its advantages as a stably virus producing cell to include (i) tight regulation by Dox; (ii) higher levels of HBV expression; (iii) two different cellular backgrounds, namely HepG2 ad Huh7; (iv) the potential, in form of the precharacterized tTA-lines, to easily generate additional
Acknowledgements
This work was supported by the EU HepBvar project (QLK2-CT-2001-00977) and in part by the Deutsche Forschungsgemeinschaft (DFG Na 154/9-1). It is part of the activities of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a Grant (LSHM-CT-2004-503359) from the Priority 1 “Life Sciences, Genomics and Biotechnology for Health” programme in the 6th Framework Programme of the EU. SD is grateful for a fellowship from the University Hospital Freiburg. The
References (45)
- et al.
Effects of extended lamivudine therapy in Asian patients with chronic hepatitis B. Asia Hepatitis Lamivudine Study Group
Gastroenterology
(2000) - et al.
Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes
Gastroenterology
(2005) - et al.
Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances
Methods Enzymol
(2000) - et al.
Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo
Hepatology
(1996) - et al.
Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy
Gastroenterology
(2004) - et al.
Anti-hepatitis B virus activity of a mixture of two monoclonal antibodies in an “inhibition in solution” assay
Hepatology
(1995) - et al.
Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells
J Hepatol
(1999) - et al.
Cell-type and donor-specific transcriptional responses to interferon-alpha. Use of customized gene arrays
J Biol Chem
(2002) - et al.
Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses
Hepatology
(2002) - et al.
Novel insights into hepatitis C virus replication and persistence
Adv Virus Res
(2004)
The hepatitis C virus replicon system: from basic research to clinical application
J Hepatol
Hepatitis B Virus
Hepatitis B virus replication: novel roles for virus–host interactions
Intervirology
Macromolecular interactions in hepatitis B virus replication and particle formation
Hepadnaviridae: the viruses and their replication
Hepatitis B virus infection – natural history and clinical consequences
N Engl J Med
Current treatment of chronic hepatitis B: benefits and limitations
Semin Liver Dis
Hepatitis B virus: old, new and future approaches to antiviral treatment
J Antimicrob Chemother
Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B
Clin Infect Dis
Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B
N Engl J Med
Looking to the future: new agents for chronic hepatitis B
Am J Gastroenterol
Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus
J Virol
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2022, Pharmacological ResearchCitation Excerpt :More importantly, nucleotides of 17.3 strain remain original at common mutational sites, making HepG2-17.3 an applicable prototype cellular tool of genotype C2 HBV study. We selected HepG2 as parental cell because HBV replication in HepG2 is more sustainable and stable than other cells [19,25]. Following the same establishment procedure, mutations could be introduced into pTRE2pur-17.3 backbone, and related mutant-HBV replication cells would contribute to perform genotype-phenotype investigation, but not limited to pharmacological study.
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The authors state that they did not receive funding from the manufacturers to carry out their research. They received funding from EU, DFG which enabled them to carry out their study.
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Present address: Department of Liver Diseases, Bethune International Peace Hospital, 050082 Shijiazhuang, PR China.