Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.

Abstract

The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another. Cells ofone clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen. HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles. Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis. Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles. This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.The human pathogen hepatitis B virus (HBV) is one of a family of small DNA hepadnaviruses that share the ability to cause liver damage but differ completely in their host range specificity. The genome of HBV (as well as those of all of the hepadnaviruses) is a relaxed circular, partially double-stranded DNA molecule that is held together by hydrogen bonding of the ='300-base-pair (bp) 5' cohesive termini (1). The (-) strand has an invariable length of -3.2 kilobases (kb), whereas the (+) strand is .20% of this length (2).Investigation of the expression and replication of the HBV genome as well as the full viral life cycle has been hampered by the lack of an in vitro tissue culture system in which HBV is propagated. In numerous attempts to rectify this situation, several mammalian cell lines have been transfected with cloned HBV DNA (3-10). Thus Knowles. These cells were transfected with pDolTHBV-1, a vector that contains two head-to-tail dimers of HBV in a tail-to-tail orientation. Transformation with the plasmid was mediated by exposure of the cells to pDolTHBV-1 in the presence of 5 umg of Polybrene per ml (17,18). The cultures were incubated at 37°C for 6 hr, and the cells then were shocked for 4 min with 30% dimethyl sulfoxide in minimal essential medium (MEM), washed several times with phosphate-buffered saline (PBS) containing Ca2' and Mg2+, and maintained thereafter in MEM supplemented with 10% fetal bovine serum and 380 ,ug of G418 per ml. Clones of cells that grew in the presence of G418 were isolated, allowed to grow to confluence, and tested for their ability to synthesize and secrete HBsAg and HBeAg. One of the clones obtained in this manner, designated 2.2.15, has been maintained for >6 months and was analyzed as described below. All cul...

Intracellular application of hairpin ribozyme genes against hepatitis B virus

“…However, it has been demonstrated that transfection of greater than full-length HBV genomes into liver cell lines will result in HBV protein expression, replication and production of infectious HBV virions. [19][20][21] These 'overlength' HBV DNA constructs mimic the circularity of the HBV genome by duplicating the HBV sequences containing the pgRNA promoter and polyA signal on both ends of a linear genome (see Figure 1a). Quantification of HBV virus particles produced in tissue culture is best achieved using the endogenous polymerase assay (EPA).…”

Cytokine and hepatitis B virus DNA Co-immunizations enhance cellular and humoral immune responses to the middle but not to the large hepatitis B virus surface antigen in mice

“…25,27,28 The mastocytoma cell line P815 (H-2 d ), the myeloma cell line SP2/0 (H-2 d ), and the mouse myoblastoma cell line G8 were obtained from the American Type Culture Collection. HepG2.2.15, 29 HepG2, 29 and HuH-7 30 cells were permanently growing in our laboratories. The HBV-LHBs stable expressing cell line P815NeopreS1 was a generous gift from Dr. F.V.…”

Ultrastructural analysis of hepatitis B virus in HepG2-transfected cells with special emphasis on subviral filament morphogenesis

“…13 By electron microscopy, HBV virions and subviral envelope filaments were found in intracellular cisternae, 12 in contrast to the absence of detectable intracellular particles in other HBV-producer-transfected cells such as the HepG2-T2.2.15 clone. 14,15 This was the consequence of the particularly high expression level of the L protein in the HepG2-T14 cells, as seen by a strong immunocytochemical staining for the preS1 antigen in these cells. 12 The HepG2-T14 clone as well as the HepG2 parent cell line were grown at 37°C with 5% CO 2 in RPMI 1640 medium (Gibco, Grand Island, NY) suppleAbbreviations: HBV, hepatitis B virus; L protein, large protein; M protein, middle protein; S protein, small protein; ER, endoplasmic reticulum; TNE, 150 mmol/L NaCl and 20 mmol/L Tris HCl, pH 7.4; FCH, fibrosing cholestatic hepatitis.…”