The hepatitis B virus as a molecular model for chronic infection: synthesis of hepatitis B surface and e antigens in mouse L cells transfected with closed circular viral DNA.
Linear hepatitis B virus (HBV) DNA, excised from a recombinant plasmid with EcoR1, was purified by preparative electrophoresis on agarose gels and incubated with phage T4 ligase to form either monomeric or dimeric closed circles. Thymidine kinase deficient mouse L cells were cotransfected with thymidine kinase (tk) and circular HBV DNAs and grown in hypoxanthine medium. Colonies of tk-transformed cells, selected after 3-4 weeks of incubation and subcultured in HAT medium, synthesized either hepatitis B surface antigen (HBsAg) alone or HBsAg in combination with hepatitis B e antigen (HBeAg). The various cell colonies differed in plating efficiency, growth rates, cellular appearance, and extent of viral antigen synthesis. Southern hybridization analysis showed the presence of HBV-related sequences in high molecular weight DNA prepared from cells expressing viral antigens. Digestion of cellular DNAs with restriction endonucleases indicated integration of the entire viral genome.
D000818:Animals; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004279:DNA, Viral / Q000276:immunology; D004587:Electrophoresis, Agar Gel; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D006511:Hepatitis B Antigens / Q000276:immunology / Q000302:isolation & purification*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007739:L Cells / Q000276:immunology; D008297:Male; D051379:Mice
D004279:DNA, Viral; D006511:Hepatitis B Antigens