Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

Abstract

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC₅₀: 1.4 nM) and interferon-α (IC₅₀: 28 IU/ml, but max. 60-80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC₅₀: 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Fig. 1

Establishment and characterization of the HepNB2.7 cell line. a Schematic representation of the HBV genome with its ORFs (arrows) and the HB2.7 subgenomic construct (red) comprising the L-/M-/S-HBsAg and HBx ORFs. b NTCP-specific western blot of deglycosylated total cell lysates of parental HepG2, HepG2-NTCP, and HepNB2.7 cells. c Uptake of ³H-taurocholate in the parental or NTCP-transduced cells (black bars). Uptake was inhibited by pre- and co-incubation with 2 µM Myrcludex B (white bars). d ELISA-based quantification of HBsAg in the supernatant of the three cell lines. e HBsAg-specific western blot of the total cell lysates

Fig. 2

HepNB2.7 cells secrete infectious progeny virus after HDV infection. a Schematic representation of the experimental layout: HepG2-NTCP or HepNB2.7 cells were inoculated with 2 IU/cell HDV, the supernatant from day 12 to 14 post infection was collected, and one-tenth of the supernatant was used for secondary infection of HuH7-NTCP cells. b Cells of primary infection (top) and secondary infection (bottom) were fixed and HDAg (red) was immunostained (scale bar: 100 µm). c For infection quantification, 16 images per condition of the immunostained cells were automatically acquired and HDAg-positive cells and nuclei were counted using ImageJ software. d HepNB2.7 cells were inoculated with HBV at 150 ge/cell in the presence or absence of 500 nM MyrB. HBeAg in the cell supernatant from day 5 to 7 post infection was quantified by ELISA

Fig. 3

Long-term infection and progeny secretion. HepNB2.7 cells were inoculated with HDV at 0.2/0.6 or 2 IU/cell. The cell supernatant was collected every third day starting from day 3 post infection until day 33. a HBsAg in the supernatant was quantified by ELISA. b Total RNA was extracted from the cell supernatant and HDV RNA was quantified by RT-qPCR. c The supernatant of the respective time points was used as an inoculum for a secondary infection on HuH7-NTCP cells. HDAg-positive cells of the secondary infection were quantified by automated image analysis

Fig. 4

Fast and reliable quantification of HDV infection by in-cell ELISA. HepNB2.7 cells seeded in a 96-well plate were inoculated with fourfold dilutions of HDV starting from 1.25 IU/cell. Cells were fixed at day 8 post infection and (a, black bars) in-cell ELISA was performed or (b, scale bar: 100 µm) immunofluorescence staining of HDAg was performed and positive cells were quantified by automated image analysis (a, white bars). The hatched line marks the in-cell ELISA signal level of the uninfected control to serve as the baseline

Fig. 5

Antiviral drug evaluation on primary and secondary infection. a Schematic representation of the experimental layout: HepNB2.7 cells seeded in 96-well plates were pre-treated with the compounds for 2 h, and then inoculated with 0.3 IU/cell HDV in the presence of the compounds. The medium was replaced with fresh compounds every 2nd/3rd day. The supernatant from day 10 to 12 post infection was collected and one-fifth of the supernatant was used as an inoculum for secondary infection of HuH7-NTCP cells. b At day 12 post primary infection, cells were fixed and HDAg was quantified by in-cell ELISA. c HDAg of the secondary infection was quantified by in-cell ELISA at day 7 post secondary infection. All in-cell ELISA data from primary and secondary infection represent the mean of three independent infection experiments. d Cytotoxicity of the compounds was assessed on HepNB2.7 cells using WST-1 assay

Fig. 6

Inhibition of prenylation leads to an enhanced innate immune activation. HepNB2.7 cells seeded in 24-well plates were pre-treated with 50× IC₅₀ concentration of the compounds for 2 h, and then inoculated with 0.5 IU/cell HDV in the presence of the compounds. The medium was replaced with fresh compounds every 2nd/3rd day. The supernatant from day 9 to 12 post infection was collected and one-fifth of the supernatant was used as an inoculum for secondary infection of HuH7-NTCP cells. a Cells of primary (top) and secondary (bottom) infection were fixed, and HDAg was immunostained (scale bar: 100 µm). b HDAg-specific western blot of the cells after primary infection treated with the indicated compounds (top). Western blot quantification of the two HDAg isoforms (bottom). Values represent the mean of three individual blots. c HepNB2.7 cells were fixed after primary infection and co-immunostained with a human serum for the total HDAg (red) and with a rabbit serum specifically for L-HDAg (green) (scale bar: 50 µm). d, e The total RNA was extracted from HepNB2.7 cells after primary infection, reverse transcribed, and the total HDV genomes, interferons, and ISGs were quantified by qPCR (n = 3 replicates). f, g, h Primary human hepatocytes (PHH) were co-infected with 0.5 IU/cell HDV and 150 ge/cell HBV and treated with 25× IC₅₀ concentration of the compounds throughout the infection. The medium was replaced with fresh compounds every 2nd/3rd day. The supernatant from day 10 to 12 post infection was collected and one-fifth of the supernatant was used as an inoculum for secondary infection of HuH7-NTCP cells (f, scale bar: 50 µm). The PHH of the primary infection at d12 were lysed and S- and L-HDAg were analysed by western blot (g). The total RNA was extracted from the PHH at d12, and the total HDV genomes, IFN-lambda, and RSAD2 copies were determined by RT-qPCR (n = 3 replicates) (h). Unpaired two-tailed Student’s t test was used for statistical analyses (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. not significant, P > 0.05)