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...
Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.
This report identifies L-ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4 mM L-ethionine, globin mRNA accumulates and in 4-5 days, 25 -30 of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine-induced accumulation of globin mRNA and er y thr oid differentiation.At the concentration where L-ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division.To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from F L cells induced to differentiate with dimethylsulfoxide and butyrate.In contrast to the tRNA from ethionine-treated cells, tRNA from cells induced by butyrate or MezSO cannot be methylated in vitro using homologous enzymes.DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.
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