Continuous Expression and Replication of the Hepatitis δ Virus Genome in Hep G2 Hepatoblastoma Cells Transfected with Cloned Viral DNA

To establish stable cell clones allowing continuous replication of hepatitis δ virus (HDV), Hep G2, a hepatoblastoma cell line containing no hepatitis B virus (HBV) DNA sequences, was transfected with a recombinant plasmid containing a tandem trimer of HDV cDNA (driven by the simian virus 40 late pr...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1990-07, Vol.87 (14), p.5253-5257
Main Authors: Chen, Pei-Jer, Kuo, Mark Yen-Pin, Chen, Mei-Ling, Tu, Su-Jen, Chiu, Mei-Na, Wu, Hui-Lin, Hsu, Hey-Chi, Chen, Ding-Shinn
Format: Article
Language:English
Subjects:
Antigens
Antigens, Viral - analysis
Antigens, Viral - genetics
Blotting, Southern
Blotting, Western
Carcinoma, Hepatocellular
Cell Line
Cell lines
Cloning, Molecular
DNA
DNA Replication
DNA, Viral - genetics
Fluorescent Antibody Technique
Genes, Viral
Genomes
Hep G2 cells
Hepatitis B virus
Hepatitis delta virus
Hepatitis Delta Virus - genetics
Hepatocytes
Humans
Liver Neoplasms
Plasmids
RNA
Transfection
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Summary:To establish stable cell clones allowing continuous replication of hepatitis δ virus (HDV), Hep G2, a hepatoblastoma cell line containing no hepatitis B virus (HBV) DNA sequences, was transfected with a recombinant plasmid containing a tandem trimer of HDV cDNA (driven by the simian virus 40 late promoter) and a neomycin-resistance gene. After selection with the neomycin analogue G418, at least two of the resistant clones were shown to have intact δ antigen by specific immunoblotting, and the δ antigen was located in the cell nucleus by immunofluorescence. Transfected cloned viral DNAs were found to be integrated into cell chromosomes. Replication of the HDV genome was demonstrated by the presence of not only genomic and antigenomic HDV RNAs but also HDV RNAs in multimeric and circular forms. In addition, a 0.8-kilobase antigenomic RNA containing a poly(A) tail and encoding the δ-antigen open reading frame was documented. Continuous replication and transcription of the HDV genome was thus achieved in these transfected cell lines. The results confirmed that replication of HDV was unassisted by HBV. Stable passage of such cell lines strongly suggests that HDV lacks direct cytopathicity in hepatocytes. These clones should be useful in studying the details of the HDV life cycle and the relationship between HDV and its helper virus, HBV.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.87.14.5253