Raney, Anneke K. (1993) Transcriptional regulation of the hepatitis B virus large surface antigen gene. PhD thesis, University of Warwick.

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Abstract

Hepatitis B virus (HBV) is a hepatotropic virus of highly restricted host range and tissue specificity. Although the mechanisms governing this tropism are not fully understood, it is likely that restrictions occur at multiple steps in the viral life cycle. The liver-specific regulation of HBV gene expression suggests that transcription may be an important factor in the hepatotropism of the virus. An analysis of tissue- or cell- line-specific regulation of the HBV promoters may elucidate the role of transcriptional regulation in the hepatotropism of the virus.
The major aim of this project was to characterize the transcriptional regulation of the large surface antigen gene of hepatitis B virus. To achieve this, the regions of the HBV genome involved in the regulation of the expression of the large surface antigen gene were identified using a transient transfection system in mammalian cell lines. The transcriptional activities of the four HBV promoters were compared in the human differentiated hepatoma cell lines Hep3B, PLC/PRF/5, HepG2 and Huh7, a human dedifferentiated hepatoma cell line HepG2.1, and the nonhepatoma cell lines HeLa S3 and NIH 3T3. To determine the relative transcriptional activities of the four HBV promoters, reporter gene plasmids were generated such that the expression of the firefly luciferase gene was under the control of each of the HBV promoters in the context of the complete genome. The nucleocapsid promoter and large surface antigen promoter displayed higher relative activities in the differentiated hepatoma cell lines, indicating that these promoters are preferentially active in these cell lines. A series of large surface antigen promoter deletion plasmids were constructed to identify the important regulatory regions of the large surface antigen promoter. The deletion analysis demonstrated that the region responsible for the high relative activity in differentiated hepatoma cell lines is located between -90 and -76 relative to the transcription initiation site (-*■!) located at map position 2809. This sequence element contains the binding site (GTTAATCATTACT) for the liver-enriched transcription factor hepatocyte nuclear factor I, HNF1. A eukaryotic expression vector containing the HNFI cDNA under the control of the mouse metallothionein I promoter was cotransfected with the HBV promoter constructs in Huh7 and HepG2.1 cells, and the relative levels of activity were determined. The Huh7 cell line was used because it is one of the cell lines in which HBV replication and particle production can occur and may represent the tissue culture system closest to the natural environment for the HBV life cycle, the liver cell. The cloned transcription factor HNFI activated transcription from the large surface antigen promoter, but not from any of the other HBV promoters. Cotransfection experiments using the HNFI cDNA expression vector and large surface antigen promoter deletion constructs demonstrated that this transactivation was mediated through the HNFI binding site located between -90 and -76 in the large surface antigen promoter. A series of deletion mutants of the cDNA in the HNFI expression vector was
generated to determine the transcriptional activation domain of the HNFI polypeptide. The major domain of the HNFI polypeptide involved in transcriptional activation of the large surface antigen promoter in the human hepatoma cell line HepG2.1 was mapped to a region rich in glutamine and proline residues (9 of 18 residues). To demonstrate directly that the HNFI polypeptide produced by the expression of the HNFI cDNA could bind the large surface antigen promoter HNFI recognition sequence, and to determine whether a protein present in the differentiated hepatoma cell line Huh7 bound the HNFI element, gel mobility shift analysis was performed. This analysis demonstrated that a protein present in nuclear extracts from Huh7 cells formed a specific complex with the HNFI binding site which had similar migration properties to the complex formed between exogenously expressed HNFI and the HNFI recognition sequence. DNase I footprinting analysis demonstrated the binding of a protein present in the differentiated hepatoma cell line Huh7 to the HNFI recognition sequence in the large surface antigen promoter. DNase I footprinting also showed that purified TATA binding protein binds the TATA box element located between -31 and -23 in the large surface antigen promoter. The analysis of synthetic promoter constructs suggested that the HNFI and TATA box elements were the only elements necessary for maximal activity from the large surface antigen promoter, and analysis of clustered point mutations in the large surface antigen minimal promoter region demonstrated that sequences between the HNFI and TATA box elements were not required for the HNF1- dependent activity of the large surface antigen promoter. These studies suggested that the liver-enriched transcription factor HNFI plays a critical role in the cell-line and tissue-specific regulation of the HBV large surface antigen promoter.

Item Type:	Thesis (PhD)
Subjects:	R Medicine > RC Internal medicine
Library of Congress Subject Headings (LCSH):	Hepatitis B virus, Hepatitis B, Hepatitis associated antigen -- Analysis, Cytodiagnosis, Genetics
Official Date:	July 1993
Dates:	Date Event July 1993 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Easton, A. J. (Andrew J.)
Extent:	vi, 160 leaves
Language:	eng

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