An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection

Abstract

Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

Figure 1. Phase-contrast and transmission electron micrographs under proliferating conditions.

(A) Morphology of Hep-Y1 and Hep-Y2 cells in monolayer culture by phase-contrast microscopy. Cells were maintained in RPMI-1640 medium. Arrows indicated granular hepatocyte-like appearance. Arrowheads indicated hepatic plate-like structure. (B) Low- magnification views of a single Hep-Y2 cell by transmission electron microscopy. Arrows indicated prominent nucleoli. Arrowheads indicated mitochondria. (C) High-magnification views of a single Hep-Y2 cell by transmission electron microscopy. Arrowheads indicated mitochondria. (D) Low- magnification views of a single Hep-Y1 cell by transmission electron microscopy. Arrows indicated prominent nucleoli. Arrowheads indicated mitochondria. (E) High-magnification views of a single Hep-Y1 cell by transmission electron microscopy. Arrowheads indicated mitochondria.

Figure 2. Standard Giemsa-banded karyotype analysis.

Representative chromosomal structures of Hep-Y1 cell (A) and Hep-Y2 cell (B) displayed aneuploidy and complicated genetic abnormalities. Arrows indicated chromosome gains. Arrowheads indicated marker chromosomes.

Figure 3. Liver-specific protein expression in culture cells and HBV DNA detection in culture medium.

(A) Expression of liver-specific proteins in Hep-Y1 and Hep-Y2 cells. Western blot analysis of albumin, transferrin, and aldolase B in Hep-Y1, Hep-Y2, HepG2, and 293-EBNA kidney cells. (B) PCR products in the medium of cultured Hep-Y1 cells and Hep-Y2 cells on a 2% agarose gel. M, molecular weight marker; P, PCR product from HBV DNA using the same PCR primers as positive control. Lanes 1 to 3, and lanes 4 to 6 were results of triplicate experiments.

Figure 4. HBV DNA replication in Hep-Y1 cells after HBV infection.

(A) Southern blot kinetic analysis of PCR products after infection of Hep-Y1 cells with control normal HBV-negative serum and HBV-containing serum. P1 and P2, 1 pg and 50 pg of PCR products derived from HBV DNA were loaded. (B) Quantification of HBV DNA. The HBV-DNA levels in 10⁶ cells after HBV infection were measured by Cobas TaqMan HBV assay. D1-3, Day 1–3 after infection. 1 IU = 5.82 copies.

Figure 5. Permissiveness of Hep-Y2 and Hep-Y1 cells for HBV infection.

(A) Southern blot kinetic analysis of cytosolic and nuclear HBV DNA after infection of Hep-Y2 cells with control normal serum and HBV-containing serum. (B) Southern blot kinetic analysis of cytosolic and nuclear HBV DNA after infection of Hep-Y1 cells with control normal serum and HBV-containing serum. C, un-infected HepG2 cells as negative control. P, 20 pg of PCR product from HBV DNA as positive hybridization control.

Figure 6. Immunofluorescence analysis of core protein expression after HBV infection.

(A) HBV-infected HepG2 and Hep-Y1 cells (left two panels) and HBV-negative serum inoculated Hep-Y1 and Hep-Y2 cells (right two panels, as negative controls) (B) HBV infected Hep-Y2 cells. Data of three independent experiments were shown (top to bottom). A polyclonal anti-HBc antibody was used to detect HBcAg. Nuclei were visualized by DAPI staining.

Figure 7. Immunofluorescence analysis of surface protein expression after HBV infection.

(A) HBV-infected HepG2, Hep-Y1, and Hep-Y2 cells. (B) HBV-negative serum inoculated Hep-Y1 and Hep-Y2 cells. A polyclonal anti-HBs antibody was used to detect HBsAg. Nuclei were visualized by DAPI staining.