Abstract
ABSTRACT Baculovirus has been employed as an efficient gene delivery vector into mammalian cells for a variety of purposes. In this study, we further expanded the applications of baculovirus as a tool to study the assembly of hepatitis delta virus-like particle. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen and hepatitis B surface antigen under the regulation of mammalian promoters. Simple and efficient gene transduction into hepatoma cell lines (80-90% as determined by flow cytometry) and high level transgene expression (in the order of microgram per million cells) were achieved by incubating the cells with unconcentrated virus supplemented with Dulbecco’s phosphate-buffered saline at 25 □C for 6 h. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus, even for cells grown to near confluency. HDV antigen expression was enhanced by EGTA pretreatment and addition of sodium butyrate in culture medium and prolonged by superinfection. L-HDAg was correctly isoprenylated and localized to nucleus of the transduced cells as the authentic protein. Upon co-transduction, HDV-like particle could be assembled and secreted into medium, which were further purified from the culture medium by isopycnic ultracentrifugation and visualized by electron microscopy. This study demonstrated that baculovirus transduction is a suitable method alternative to plasmid transfection for efficient formation of HDV-like particles. This system may be also a useful tool in investigating cellular processes involved in HDV assembly and in producing large amount of HDV-like particles in bioreactor for medical research.