Expression of cloned hepatitis B virus DNA in human cell cultures.

Abstract

DNA was isolated from the ayw subtype of hepatitis B virus (HBV) that was incubated in vitro with all 4 deoxynucleoside triphosphates to complete the circular viral genome by 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 [human cervical carcinoma] 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. EM 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 DNA evoked these effects in HeLa cells, both cytophathic changes and intranuclear hepatitis B core antigen were detected in HeLa cells infected with Dane particles.