A single hepatitis B virus genome with a reporter allows the entire viral life cycle to be monitored in primary human hepatocytes

Abstract

For the development of antiviral agents to eliminate hepatitis B virus (HBV), it is essential to establish an HBV cell culture system that can easily monitor HBV infection. Here, we created a novel HBV infection monitoring system using a luminescent 11‐amino acid reporter, the high‐affinity subunit of nano‐luciferase binary technology (HiBiT). The HiBiT‐coding sequence was inserted at the N‐terminus of preS1 in a 1.2‐fold plasmid encoding a genotype C HBV genome. After transfection of HepG2 cells with this HiBiT‐containing plasmid, the supernatant was used to prepare a recombinant cell culture‐derived virus (HiBiT‐HBVcc). Primary human hepatocytes (PXB) were inoculated with HiBiT‐HBVcc. Following inoculation, intracellular and extracellular HiBiT activity and the levels of various HBV markers were determined. Reinfection of naive PXB cells with HiBiT‐HBVcc prepared from HiBiT‐HBVcc‐infected PXB cells was analyzed. When PXB cells were infected with HiBiT‐HBVcc at several titers, extracellular HiBiT activity was detected in a viral titer‐dependent manner and was correlated with intracellular HiBiT activity. Inhibitors of HBV entry or replication suppressed extracellular HiBiT activity. Viral DNA, RNA, and proteins were detectable, including covalently closed circular DNA, by Southern blot analysis. The synthesis of relaxed‐circular DNA from single‐stranded DNA in HiBiT‐HBV decreased to one third of that of wild‐type HBV, and the infectivity of HiBiT‐HBVcc decreased to one tenth of that of wild‐type HBVcc. HiBiT‐HBVcc prepared from PXB cells harboring HiBiT‐HBV was able to infect naive PXB cells. Conclusions : Recombinant HiBiT‐HBV can undergo the entire viral life cycle, thus facilitating high‐throughput screening for HBV infection in vitro using supernatants. This system will be a powerful tool for developing antiviral agents.

Functional involvement of endothelial lipase in hepatitis B virus infection

“…For this assay, we used a recombinant cell culture-derived virus labeled with a luminescent 11-amino acid reporter (HiBiT-HBVcc) (Figure 5 A). 12 HSPGs in cells were captured by an anti-heparin sulfate antibody that was immobilized on the plate, and then HiBiT-HBVcc was added for binding. As the source of HSPGs, lysates of Huh7-NTCP-YFP cells were used, and significant luminescent signals were observed (Figure 5 B).…”

“…Recently, we have reported a novel HBV infection monitoring system using a luminescent 11-amino acid reporter, the high-affinity subunit of nano-luciferase binary technology (HiBiT), cloned upstream of a genotype C HBV genome, to generate recombinant cell culture-derived virus (HiBiT-HBVcc). 12 Whole cell lysates of Huh7-NTCP-YFP and HepG2-NTCP-C4 Lenti-Ctrl or Lenti-LIPG cells were added to the plate of a Human Heparan Sulfate ELISA Kit (Elabscience, Houston, TX), and incubated for 90 minutes at 37°C with or without recombinant human LIPG protein (Abcam). After incubation, the plate was washed 3 times with wash buffer.…”