Generation, characterization, and use of the HBV baculovirus-HepG2 system: A novel in vitro model system for studying hepatitis B virus

Baculovirus-mediated gene transfer has been further investigated in primary rat hepatocytes, SV40-immortalized rat hepatocyte cell lines, and human hepatic cell lines. Infection of primary hepatocytes in short-term culture with CMV-lacZ, a recombinant baculovirus carrying the lacZ gene under the control of the CMV promoter, resulted in a 10-fold increase in transfection efficiencies compared to classical transfection methods. Infection of primary hepatocytes in long-term culture with CMV-lacZ baculovirus resulted in efficient transfection of cells on hepatocyte island peripheries; however, cells on island interiors were poorly transfected. SV40-immortalized rat hepatocyte cell lines and human hepatic cell lines were highly susceptible to baculovirus-mediated gene transfer.; A baculovirus encoding a hepatitis B virus (HBV) genome driven only by endogenous HBV promoters was generated. Infection of HepG2 cells with HBV baculovirus resulted in efficient expression of HBV transcripts and antigens. HBV replication occurred as evidenced by high levels of replicative intermediates, extracellular protected HBV DNA with a density identical to Dane particles, and CCC HBV DNA. Characterization of the HBV baculovirus-HepG2 cell system indicated: (1){A0}a wide range in levels of HBV replication can be obtained by changing the moi; (2){A0}replication levels exceeding those of conventional HBV-expressing cell lines are easily produced; (3){A0}the transient nature of HBV expression can be extended by superinfecting cultures with HBV baculovirus; (4){A0}cultures can be manipulated and/or treated before or after the initiation of HBV expression; (5){A0}high levels of replication allow rapid detection of HBV products including CCC HBV DNA; and (6){A0}baculovirus infection is not toxic to HepG2 cells.; The HBV baculovirus-HepG2 system was used to further investigate 3TC, a nucleoside analog previously shown to interfere with HBV replication. Treatment of HBV baculovirus-infected HepG2 cells resulted in a dose and time dependent inhibition of HBV replication. Observed reductions in extracellular HBV DNA after 3TC treatment agreed well with previous reports. Importantly, this system made it possible to observe that 3TC treatment causes a large reduction in CCC HBV DNA levels in infected cells. These studies indicate that the HBV baculovirus-HepG2 system can be used to complement existing in vitro models used for testing antiviral compounds.