Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter.
We have demonstrated previously the presence of an 8-bp deletion mutant, spanning from nt. 1768 to nt. 1775 in the basic core promoter region of hepatitis B virus (HBV) in patients with anti-HBe positive asymptomatic phase before developing acute exacerbation after immunosuppressive treatment. The transcription and progeny virus production activities of the mutant were examined by transfection of the recombinant plasmid [pUC Del(2)] containing the head-to-tail dimer DNA of the mutant into HepG2 cells. The amounts of hepatitis B surface antigen (HBsAg) and HBe antigens secreted into the culture medium were markedly reduced. Southern blotting of DNAs extracted from the culture medium also showed reduced mutant activity to produce progeny virus. Northern blotting and RNase protection assay of RNAs extracted from transfected cells demonstrated that the transcription of both precore mRNA and pregenome RNA was reduced significantly compared to that of wild-type HBV. The promoter activity examined by transfection of the CAT plasmid containing deletion mutant DNA was much lower than that of wild type. Co-transfection experiments, however, of the CAT plasmid containing wild-type DNA with pUC Del(2) reduced CAT activity induced by wild-type, suggesting that truncated X protein produced by the mutant does not possess a sufficient transactivating activity. Gel shift assay using HepG2 nuclear extract and a probe containing four TA-rich regions in CP and various competitors suggested that the lack of the third TA-rich region was responsible for the transcription reduction of precore mRNA and pregenome RNA. The possible mechanisms are discussed.
Kohno
K
K
Department of Microbiology, Oita Medical University, Oita, Japan.
Nishizono
A
A
Terao
H
H
Hiraga
M
M
Mifune
K
K
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D006511:Hepatitis B Antigens / Q000096:biosynthesis; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D006511:Hepatitis B Antigens; D009687:Nuclear Proteins