Expression of cloned hepatitis B virus DNA in human cell cultures.
DNA was isolated from the ayw subtype of hepatitis B virus (HBV) that had been incubated in vitro with all four deoxynucleoside triphosphates in order to complete the circular viral genome by means of the endogenous DNA polymerase. The purified viral DNA was cleaved with EcoRI restriction endonuclease, inserted into the EcoRI site of plasmid pBR322, and cloned in Escherichia coli chi 1776. DNA from a clone, pHBV-1, that contained a 3200-base-pair insert of HBV DNA was cleaved with EcoRI and incubated with phage T4 ligase under conditions favoring intramolecular ligation. HeLa cell cultures exposed to this DNA showed marked cytopathic changes, accompanied by production of hepatitis B core and surface antigens, 11-14 days after subculture. Electron microscopic examination of anti-hepatitis B surface antigen immunoprecipitates from culture media of these cells revealed both 42-nm particles with central cores and 20-nm round particles. Although neither intact circular nor EcoRI-cleaved linear pHBV-1 DNAs evoked these effects in HeLa cells, both cytopathic changes and intranuclear hepatitis B core antigen were detected in HeLa cells infected with Dane particles.
D003001:Cloning, Molecular; D003588:Cytopathogenic Effect, Viral; D004274:DNA, Recombinant / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D006367:HeLa Cells; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis*; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis*; D006515:Hepatitis B virus / Q000235:genetics* / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D010957:Plasmids
D004274:DNA, Recombinant; D004279:DNA, Viral; D006512:Hepatitis B Core Antigens; D006514:Hepatitis B Surface Antigens