Infection of a human hepatoma cell line by hepatitis B virus

Abstract

Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.

Fig 1.

Morphology and karyotype of HepaRG cells. Phase contrast micrographs of HepaRG cells under proliferating conditions (A), maintained in culture for 30 days without DMSO (B), and maintained for 15 days without DMSO, then treated with 2% DMSO for 15 days (C). Hepatocyte-like cells and epithelium-like cells are indicated, respectively, by “h” and “e.” A bile canaliculus is indicated by a white triangle. (Bars = 50 μm.) Electron micrographs of HepaRG cells: low magnification view of HepaRG cells (D) and higher magnification views (E and F), showing a typical bile canaliculus-like structure and glycogen accumulation, respectively. (Bars = 2 μm.) RHG banding karyotype of a representative pseudodiploid metaphase of HepaRG cells (G). A karyotype of 46<2n>,XX,+del (7)(q11;q21)inv?(7)(q21q36),-der(22)t(12;22)(p11;q11) was interpreted. The two main anomalies are indicated by arrowheads.

Fig 2.

Expression of liver-specific functions in HepaRG cells. (A) Northern blot analysis of liver-specific messenger RNA expression in two liver biopsy specimens, HepG2 cells, and HepaRG cells. Cultured cells were maintained either at confluence for 1 month (no indication) or under proliferating conditions (prolif.). A 2% DMSO treatment was performed for the last 15 days of the culture when indicated (+D). (B) Effect of inducers on CYP1A (phenacetine deethylase) and CYP3A (nifedipine oxidase) activities. The activities were measured after 72 h of treatment and are expressed as the ratio of treated cells versus corresponding control cells. The inducer concentrations (μM) are indicated below the graph.

Fig 3.

HBV infection of HepaRG cells. (A) Southern blot kinetic analysis of intracellular HBV DNA after infection of HepaRG cells. Half of the DNA extracted from 10⁶ infected cells was analyzed on a 1.5% agarose gel. The positions of the relaxed circular form (RC), covalently closed circular DNA (CCC), and single-stranded DNA (SS) are indicated on the right. Lane M corresponds to 3.5 pg of 3.2- and 1.5-kb HBV DNA restriction fragments (3.4 and 6.8 × 10⁶ molecules, respectively). Autoradiographic exposure time was 24 h. (B) HBV DNA quantification by dot blot. Samples analyzed on Fig. 3A were quantified by comparison of the hybridization signal obtained from serial dilutions of known amounts of linear double-stranded HBV DNA. On the left is indicated the amount of standard HBV DNA spotted on the filter; on the right is the calculated HBV DNA copy number expressed in genome equivalents per cell (Geq/cell). Autoradiographic exposure time was 24 h. (C) Southern blot kinetic analysis of nonprotein-bound intracellular HBV DNA after infection of HepaRG cells. Half of the DNA extracted from 10⁶ infected cells was analyzed on a 1.5% agarose gel. Positions of the relaxed circular form (RC) and covalently closed circular DNA (CCC) are indicated on the right. Lane M corresponds to 3.5 pg of 3.2- and 1.5-kb HBV DNA restriction fragments (3.4 and 6.8 × 10⁶ molecules, respectively). Autoradiographic exposure time was 96 h. (D) Northern blot kinetic analysis of HBV RNA after infection of HepaRG cells. Half of the RNA extracted from 10⁶ infected cells was analyzed on a 1.5% agarose gel. RNA sizes are indicated on the right (kb). Autoradiographic exposure time was 36 h. (E) Comparison of HBV RNA levels after infection of HepaRG cells in the presence or absence of PEG by Northern blot analysis. RNA were extracted from mock-infected cells or cells infected in the absence (−PEG) or in the presence (+PEG) of PEG. Cells were infected at a multiplicity of infection of 200, 400, 800, or 1,600 genome equivalents per cell (Geq/cell). Half of the RNA extracted from 10⁶ infected cells was analyzed on a 1.5% agarose gel. Lane M corresponds to 3.5 pg of denatured 3.2- and 1.5-kb HBV DNA restriction fragments. RNA sizes are indicated on the right. The relative amounts of subgenomic RNA detected in infected cells are indicated below the figure. Autoradiographic exposure time was 96 h. (F) Kinetic analysis of HBsAg secretion in the supernatant of infected cells. Supernatants were collected during 15 days after infection of HepaRG cells (▪) or HepG2 cells (). (G) Southern blot kinetic analysis of extracellular HBV DNA in the supernatant of infected HepaRG cells. Complete viral particles were immunoprecipitated with an anti-HBsAg antibody from 1 ml of the supernatant of infected HepaRG cells. The viral DNA was extracted, and half of the preparation was analyzed by the Southern blot procedure. Lane M corresponds to 3.5 pg of 3.2- and 1.5-kb HBV DNA restriction fragments (3.4 and 6.8 × 10⁶ molecules, respectively). Autoradiographic exposure time was 96 h.

Fig 4.

Specificity of the HBV infection. (A) Immunostaining of infected HepaRG cells for HBV core antigen. Cells were stained with an anticore antibody by immunofluorescence. Positive cells are indicated by a white arrow. (B) In vitro neutralization assay. Viral particles were incubated with serial dilutions of a monoclonal anti-HBs antibody (S 39–10; ) or an unrelated monoclonal antibody (▪) and evaluated for their infectivity on HepaRG cells. Infectivity was evaluated by measuring the HBsAg secretion in the supernatant of infected cells at day 10 pi. (C) Peptide competition of HBV infection. HepaRG cells were preincubated with different concentrations (nM) of an HBV large protein-derived peptide (AA 2–78) bearing a myristic acid (myr) on gly 2 or, as a control, with a corresponding myristoylated duck hepatitis B virus-derived peptide (AA 2–41; C). The inoculum was then added for 20 h, without removing the peptide. Infectivity was evaluated by analyzing intracellular viral RNA at 10 days pi. Half of the RNA extracted from 10⁶ infected cells was analyzed on a 1.5% agarose gel. Lane M corresponds to 3.5 pg of denatured 3.2- and 1.5-kb HBV DNA restriction fragments. Viral RNA sizes are indicated on the right. Autoradiographic exposure time was 48 h. (D) Efficiency of HBV infection and HepaRG cell differentiation. Northern blot analysis of CYP 3A4 (Upper) and HBV (Lower) RNAs after infection of HepaRG cells cultivated in presence of increasing concentrations of DMSO. RNA was extracted 10 days pi. Viral RNA sizes are indicated on the right.