Establishment and characterization of a new cell culture system for hepatitis B virus replication and infection

Abstract

Hepatitis B virus (HBV) is a primary cause of chronic liver diseases in humans. HBV infection exhibits strict host and tissue tropism. HBV core promoter (Cp) drives transcription of pregenomic RNA (pgRNA) and plays a key role in the viral life cycle. Hepatocyte nuclear factor 4α (HNF4α) acts as a major transcriptional factor that stimulates Cp. In this work, we reported that BEL7404 ​cell line displayed a high efficiency of DNA transfection and high levels of HBV antigen expression after transfection of HBV replicons without prominent viral replication. The introduction of exogenous HNF4α and human sodium taurocholate cotransporting polypeptide (hNTCP) expression into BEL7404 made it permissive for HBV replication and susceptible to HBV infection. BEL7404-derived cell lines with induced HBV permissiveness and susceptibility were constructed by stable co-transfection of hNTCP and Tet-inducible HNF4α followed by limiting dilution cloning. HBV replication in such cells was sensitive to inhibition by nucleotide analog tenofovir, while the infection was inhibited by HBV entry inhibitors. This cell culture system provides a new and additional tool for the study of HBV replication and infection as well as the characterization of antiviral agents.

Keywords: BEL7404; Doxycycline (DOX); Hepatitis B virus (HBV); Hepatocyte nuclear factor 4α (HNF4α); Sodium-taurocholate cotransporting polypeptide (NTCP); Tetracycline (Tet)-On.

Fig. 1

BEL7404 cells are not permissive for canonical HBV replication. (A) Huh7 and BEL7404 ​cells in 6-well plate were transfected with 2 ​μg pCMV-EGFP plasmid and EGFP expression at 48 ​h was visualized using fluorescent microscopy (left). Percentage of EGFP-positive cells was analyzed using imageJ software. Data are presented as means ​± ​the SD (n ​= ​10). B, C Huh7 and BEL7404 ​cells in 6-cm dishes were transfected with 4 ​μg HBV replicon constructs as indicated. At 96 ​h post-transfection, HBsAg and HBeAg in the supernatants were determined using ELISA. Data are presented as means ​± ​the SD (n ​= ​3). Dotted lines represent cut-off thresholds (B). Intracellular capsid-associated HBV DNA was analyzed using Southern blot (C). Positions of relaxed circular (RC) and replication intermediates (RI) are indicated. Transfections were performed in triplicates and repeated twice independently.

Fig. 2

Exogenous HNF4α rescues canonical HBV replication in BEL7404 ​cells. A Huh7 and BEL7404 ​cells in 24-well plates were transfected with indicated HBV promoter Firefly luciferase reporter plasmid and Renilla luciferase plasmid control. At 48 ​h post-transfection, cells were lysed and lysates were analyzed using dual-luciferase reporter assay. Means and SD of relative luciferase activities normalized against promoter-less pGL3-basic vector were plotted (n ​= ​3). B Expression of endogenous HNF1α and HNF4α in Huh7 and BEL7404 ​cells was analyzed using Western blot. C, D BEL7404 ​cells in 24-well plate were co-transfected with Cp, Sp1, and Sp2 Firefly luciferase reporter, increasing amounts of pCMV-HNF4α plasmid, and Renilla luciferase plasmid control. Promoter activities were analyzed as described above. Means and SD of relative luciferase activities normalized against empty expression vector are plotted (n ​= ​3). E, F Huh7, BEL7404 and HeLa cells in 6-cm dishes were transfected with 4 ​μg HBV replicon plasmid as indicated, with or without co-transfection of 2 ​μg pCMV-HNF4α. At 96 ​h post-transfection, HBsAg and HBeAg in the supernatants were determined using ELISA (E). Dotted lines represent cut-off thresholds. Data are presented as means ​± ​the SD (n ​= ​3). Intracellular capsid-associated HBV DNA was analyzed using Southern blot (F). Positions of relaxed circular (RC) and replication intermediates (RI) are indicated. Transfections were performed in triplicates and repeated twice independently.

Fig. 3

BEL7404-derived cells with stable hNTCP and inducible HNF4α expression support HBV replication and infection. A Recombinant lentiviral virus expressing hNTCP (left) was used to transduce BEL7404 ​cells and transductants (BEL7404-hNTCP) were selected and maintained in blasticidin-containing media. Relative levels of hNTCP mRNA and protein in parental and transduced cells were analyzed using RT-qPCR (middle) and Western blot (right). Data are presented as means ​± ​the SD (n ​= ​3). B Recombinant lentiviral viruses expressing Tet-on regulator and Tet-responsive element (TRE) controlled HNF4α were used to transduce BEL7404-hNTCP cells and triple transductants (7404NT-HNF4α) were selected and maintained in media containing blasticidin, puromycin and G418. Relative levels of HNF4α mRNA and protein in un-induced and doxycycline (DOX)-induced 7404NT-HNF4α cells were analyzed using RT-qPCR (middle) and Western blot (right). C-E 7404NT-HNF4α cells in 6-cm dishes were transfected with p1.3 ​× ​B200 and cultured with or without DOX. At 96 ​h post-transfection, intracellular HBV RNA (C), capsid-associated DNA (D) and secreted HBsAg and HBeAg (E) were analyzed using Northern blot, Southern blot and commercial quantitative assay, respectively. F 7404NT-HNF4α cells were infected by HBV (MOI ​= ​1000) in the presence of 2.5% DMSO and 4% PEG8000 for 12 ​h and cultured with or without DOX. Culture media collected at the indicated days post-infection (dpi) were analyzed for HBeAg using ELISA. Dotted lines represent cut-off thresholds. Data are presented as means ​± ​the SD (n ​= ​3). Transfections were performed in triplicates and repeated twice independently.

Fig. 4

Characterization of 7404NT-HNF4α-derived clonal cell lines established through limiting dilution. A HepG2-NTCP, 7404NT-HNF4α and 7404NT-HNF4α-derived clonal B7 and G8 cells cultured in 6-well plate were transfected with 2 ​μg pCMV-EGFP plasmid and EGFP expression at 48 ​h was visualized using fluorescent microscopy (left). Percentage of EGFP-positive cells was analyzed using imageJ software. Transfections were performed in triplicates and repeated twice independently. Data are presented as means ​± ​the SD (n ​= ​10). B Expression of hNTCP in HepG2-NTCP, BEL7404, 7404-NTCP, 7404NT-HNF4α, B7, G8 cells and the expression of HNF4α in 7404NT-HNF4α, B7 and G8 cells cultured with and without DOX were analyzed using Western blot. C Expression of ALPL, GSTM1, KYNU, HKDC1 and β-actin in Huh7, HepG2, HeLa, BEL7404, B7 and G8 cells was analyzed using Western blot.

Fig. 5

DOX-dependent HBV transcription and replication in B7 and G8 cells. A, B B7 and G8 cells in 6-well plates were transfected with p1.3 ​× ​B200 and cultured with or without DOX. At 96 ​h post-transfection, secreted HBsAg A and HBeAg B, intracellular HBV RNA C, capsid-associated DNA D were analyzed using commercial quantitative assay, Northern blot and Southern blot, respectively. Data are presented as means ​± ​the SD (n ​= ​3). Transfections were performed in triplicates and repeated twice independently.

Fig. 6

DOX-dependent HBV transcription and replication in HBV-infected B7 cells. A-D B7 cells were infected with HBV (MOI ​= ​1000) and cultured with or without DOX. Secreted HBeAg was analyzed using ELISA. Intracellular capsid-associated HBV DNA, pgRNA and nuclear cccDNA were analyzed using by qPCR or RT-qPCR. E DMSO and PEG requirement for HBV infection of B7 cells. HepG2-NTCP and B7 cells were infected with HBV (MOI ​= ​1000) in the presence of indicated DMSO and/or PEG8000 for 12 ​h. Secreted HBeAg was analyzed using ELISA. F HepG2-NTCP and B7 cells were infected with HBV (MOI ​= ​1000) in the presence of indicated DMSO and PEG8000 for 12 ​h. Nuclear cccDNA were analyzed using qPCR. Dotted lines represent cut-off thresholds. Data are presented as means or means ​± ​the SD (n ​= ​3).

Fig. 7

Evaluation of HBV-targeting agents using B7 cells. A B7 cells were transfected with p1.3 ​× ​B200 and cultured in DOX-containing media with or without 2 ​μmol/L Tenofovir disoproxil fumarate (TDF). Intracellular capsid-associated HBV DNA was analyzed using Southern blot. Transfections were performed in triplicates and repeated twice independently. B B7 cells were infected with HBV and cultured in DOX-containing media with or without 2 ​μmol/L TDF. Intracellular capsid-associated HBV DNA in cells harvested at indicated time points was analyzed using qPCR. C-E B7 cells were infected with HBV in the presence of 2 ​μmol/L Myrcludex-B or 2 ​μg/mL anti-HBs monoclonal antibody G12 (simplified as G12). Secreted HBeAg was analyzed using ELISA. Intracellular pgRNA and capsid-associated HBV DNA were analyzed using by qPCR. Data are presented as means or means ​± ​the SD (n ​= ​3). Dotted lines represent cut-off thresholds.