Identification of integrated hepatitis B virus DNA and expression of viral RNA in an HBsAg-producing human hepatocellular carcinoma cell line.

Abstract

Alexander and co-workers1 have developed a tissue culture cell line (PLC/PRF/5) from a primary hepatocellular carcinoma of a Mozambican male with a positive serum for hepatitis B virus surface antigen (HBsAg). This cell line produces and secretes HBsAg but no other known viral protein1–3. PLC/PRF/5 therefore contains at least part of the hepatitis B virus (HBV) genome in functional form. The 42-nm, spherical Dane particle in human serum is thought to represent the complete infectious agent of HBV4. The core of this particle contains circular double-stranded DNA in which one strand is ‘nicked’ (complete but not covalently closed) and the other strand is incomplete (lacking 15–45% of its sequence complement)4,5. Four proteins have been associated with the HBsAg, core antigen (HBcAg), e antigen and a viral DNA polymerase4. Several groups have now cloned Dane particle DNA in Escherichia coli6–10, and both restriction maps and a complete DNA sequence have been determined. Various studies suggest that there is sequence heterogeneity in Dane particle DNA isolated from patients expressing the same or different viral subtypes, as well as sequence variation in different clones of Dane particle DNA obtained from a given individual10,11. Other studies12,13 have reported HBV DNA sequences in DNA from liver cancer tissue. In none of these cases was viral DNA reported to be integrated into the host cell genome. With the improvement in techniques for characterizing viral DNA molecules, we have begun to analyse HBV genes and their expression in the PLC/PRF/5 cell line by molecular hybridization of cellular DNA and RNA to cloned 32P-labelled plasmid HBV DNA. We now present evidence in PLC/PRF/5 cells for integration of HBV DNA into the host genome and expression of three RNA molecules containing HBV-specific sequences. These findings are consistent with observations in several animal virus models in which integrated viral DNA is found during oncogenic transformation.