Investigating the hepatitis B virus life cycle using engineered reporter hepatitis B viruses

Abstract

Chronic infection with hepatitis B virus (HBV) increases the risk of developing fibrosis, cirrhosis or hepatocellular carcinoma. Current therapies are limited to type-I interferons and/or nucleos(t)ide analogues; however, these are only partially effective. The development of novel anti-HBV agents for new treatment strategies has been hampered by the lack of a suitable system that allows the in vitro replication of HBV. Studies of virus infection/replication at the molecular level using wild-type HBV are labor-intensive and time-consuming. To overcome these problems, we previously constructed a recombinant reporter HBV bearing the NanoLuc gene and showed its usefulness in identifying factors that affect HBV proliferation. Because this system mimics the early stage of the HBV life cycle faithfully, we conducted a quantitative analysis of HBV infectivity to several human hepatocyte cell lines as well as the effect of dimethyl sulfoxide and HBV protein X on the early stage of HBV proliferation using this system. Furthermore, we developed a system to produce a reporter HBV expressing a pol gene. These reporter HBV may provide an opportunity to enhance our understanding of the HBV life cycle and aid strategies for the development of new anti-HBV agents.

Keywords: anti- hepatitis B virus agents; hepatitis B virus; replication; reporter gene; reporter hepatitis B virus.

Figure 1

Schematic diagram of reporter hepatitis B virus generated in this study. pgRNA contains 3363 nucleotides. Nucleotide sequences of seven bases at both ends in pgRNA are shown. Open reading frames shown with blue rectangles indicate nucleotide numbers of the initiation and the C‐terminal in each protein. Red rectangles indicate the NanoLuc gene or secNL gene with several flanking nucleotide sequences. GLuc gene containing DNA is shown as a green rectangle. Dotted lines indicate deleted sequences in the pgRNA. Insertion site of the reporter genes is between 223 and 224 of pgRNA. Nucleotide numbers of the 5′ and 3′ end of the deleted sequences in pgRNA are also shown. Numbers in italics represent the DNA size of the inserted genes. Numbers on the right indicate the size of the recombinant pgRNA

Figure 2

Analysis of human hepatocyte‐derived cell lines and HeLa cells for hepatitis B virus/NanoLuc (HBV/NL) infectivity. HeLa, HepG2, HuH7 and PLC/PRF/5 were obtained from ATCC. PH5CH and HuS cells are described elsewhere.23, 24 (A) Each cell line (−) and NTCP transduced cell line (+) was infected with HBV/NL, and NL activity was measured 6 days after infection. (B) A single cell clone of NTCP‐myc gene transduced HepG2 cells was obtained by end‐point dilution. After 6 days of HBV/NL infection to each cell clone, NL activity was measured. (C) Western blot of NTCP‐myc using the anti‐Myc antibody and of β‐actin. Plural bands reflect modification in NTCP. (D) Infection of HBV to NTCP‐myc transduced HepG2 and HuH7 as well as western blot to detect expression of NTCP‐myc. At 6 days after infection, HBV RNA in cell lysates was measured by RT‐PCR. Detection of NTCP‐myc was conducted using anti‐Myc antibody

Figure 3

Effect of DMSO on hepatitis B virus/NanoLuc (HBV/NL) infection. The experimental protocol is shown at the top. HBV/NL was used to infect cells for 24 h in the presence of 4% PEG with or without 2% DMSO. Then, the medium was changed to new medium with or without 2% DMSO in the presence of PEG. HepG2 and HuH7 cells were used as a negative control

Figure 4

Effect of hepatitis B virus (HBV) X gene expression on the NanoLuc (NL) activity of cells infected with HBV/NL and its derivatives. (A) Amino acid in the protein X open reading frame (ORF) in the HBV/NL genome was mutated as described in the Materials and Methods. Putative ORF in the mutated X gene are shown in blue. (B) Suppressed NL activity of HBV/NL derivatives in infected cells was enhanced by ectopic expression of the HBV X gene. Viruses were used to infect HepG2/NTCP#22 cells (−) or HepG2/NTCP#22 cells transfected with pCAG X 1 day before infection (+). NL activity was measured at day 6 after infection. Western blot of HBV X and β‐actin is shown

Figure 5

Comparative analysis of the RNA level of reporter virus in cells infected with hepatitis B virus/NanoLuc (HBV/NL), HBV/secNL and HBV/GLuc as well as analysis of the correlation of reporter activity with virus RNA. (A) Relative HBV RNA level in cells infected with HBV/NL, HBV/secNL and HBV/GLuc. Recombinant viruses equivalent to 100 DNA copies per cell were infected to 1 × 10⁶ HepG2/NTCP#22 cells. RNA in cell lysates after 6 days of infection were analyzed for HBV RNA by quantitative RT‐PCR. (B,C) Time course‐dependent measurement of the relative amount of reporter activity and RNA of the reporter HBV in infected cells. One hundred copies of HBV/secNL (B) or HBV/GLuc (C) per cell were used to infect 1 × 10⁶ HepG2/NTCP#22 cells. At the indicated days, culture medium of HBV/secNL infected cells or HBV/GLuc infected cells were measured for reporter activity. Simultaneously, virus RNA in a 1/10 volume of cell lysate was quantitated by RT‐PCR using the primer set described in the Materials and Methods

Figure 6

Comparative analysis of virus RNA in transfected cells and the amount of viruses produced from those cells. (A) Six days after transfection, 30 μg RNA from wild pHBV, pHBV/NL and pHBV/NL(S+pol)‐transfected cells was analyzed by northern blot using digoxigenin (DIG) HBV RNA probes covering the full‐length HBV genome. The gel was stained with ethidium bromide to visualize ribosomal RNA. The efficiency of transfection was similar (data not shown). (B,C) Six days after transfection by wild pHBV, pHBV/NL and pHBV/NL(S+pol), virus fractions from culture medium were harvested. One‐tenth of the volume of culture medium was used to infect HepG2/NTCP#22. NL activity and the amount of virus RNA in cell lysates in (B) and (C), respectively, were measured after 6 days of infection. HBV, hepatitis B virus; NL, NanoLuc

Figure 7

Production of reporter hepatitis B virus (HBV) by co‐transfecting pHBV/D or a helper HBV lacking polymerase activity. HepG2 cells were transfected with pHBV/NL (A), pHBV/NLS+pol (B) and pHBV/NLS+polS (C) with respective helper plasmids that expressed the entire virus proteins (pHBV/D), or expressed all proteins except pol (pHBV/MDH and pHBV/MHD). Six days after transfection, the virus fraction was harvested and the same volume of virus was used to infect HepG2/NTCP#22 cells. Six days after infection, NL activity was analyzed and indicated by relative NanoLuc activity. NL, NanoLuc

Figure 8

Trans‐rescue experiment of reporter hepatitis B virus (HBV) production. Reporter viruses were harvested from HepG2 transfected with the indicated plasmids together with pHBV/D. The viruses were used to infect 2 × 10⁵ PXB cells in the presence or absence of 100 copies per cell of HBV obtained from primary human hepatocytes (PHH) maintained in urokinase‐type plasminogen activator transgenic/SCID mice. Virus was prepared from the medium and used to infect 1 × 10⁵ HuH7/NTCP cells. NanoLuc (NL) activity was measured at the indicated times. On day 16, an aliquot of the cells was treated with 80 nmol/L entecavir. Solid lines indicate relative NL activity in HuH7/NTCP infected with culture medium recovered from PXB cells infected with HBV/NL (red), HBV/NLS+pol (green) and HBV/NLS+polS (blue) together with wild‐type HBV. Dotted lines indicate relative NL activity in cells infected with culture medium recovered from PXB cells infected with HBV/NL (red), HBV/NLS+pol (green) and HBV/NLS+polS (blue) without wild‐type HBV. Double lines with red (HBV/NL) and with green (HBV/NLS+pol) show NL activity after entecavir treatment. Experiments were conducted twice and the mean value is plotted in this figure