Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.
A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.
D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D005814:Genes, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000648:ultrastructure; D006801:Humans; D008113:Liver Neoplasms; D008854:Microscopy, Electron; D014158:Transcription, Genetic; D014162:Transfection
D004279:DNA, Viral