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Journal of Virology, February 2000, p. 1241-1251, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Multiple Transcription Factors, HLF, FTF, and E4BP4, Controlling Hepatitis B Virus Enhancer II

Hisashi Ishida,1 Keiji Ueda,2 Kazuyoshi Ohkawa,1 Yoshiyuki Kanazawa,1 Atsushi Hosui,1 Fumihiko Nakanishi,1 Eiji Mita,1 Akinori Kasahara,3 Yutaka Sasaki,4 Masatsugu Hori,1 and Norio Hayashi4,*

Department of Internal Medicine and Therapeutics,1 Department of Microbiology,2 Department of General Medicine,3 and Department of Molecular Therapeutics,4 Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan

Received 28 May 1999/Accepted 3 November 1999

Hepatitis B virus (HBV) enhancer II (EnII) is a hepatotropic cis element which is responsible for the hepatocyte-specific gene expression of HBV. Multiple transcription factors have been demonstrated to interact with this region. In this study, the region from HBV nucleotides (nt) 1640 to 1663 in EnII was demonstrated to be essential for enhancer activity and to be another target sequence of putative transcription factors. To elucidate the factors which bind to this region, we used a yeast one-hybrid screening system and cloned three transcription factors, HLF, FTF, and E4BP4, from a human adult liver cDNA library. All of these factors had binding affinity to the sequence from nt 1640 to 1663. Investigation of the effects of these factors on transcriptional regulation revealed that HLF and FTF had stimulatory activity on nt 1640 to 1663, whereas E4BP4 had a suppressing effect. FTF coordinately activated both 3.5-kb RNA and 2.4/2.1-kb RNA transcription in a transient transfection assay with an HBV expression vector. HLF, however, activated only 3.5-kb RNA transcription, and in primer extension analysis, HLF strongly stimulated the synthesis of pregenome RNA compared to precore RNA. Thus, FTF stimulated the activity of the second enhancer, while HLF stimulated the activity of the core upstream regulatory sequence, which affects only the core promoter, and had a dominant effect on the pregenome RNA synthesis.

* Corresponding author. Mailing address: Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3441. Fax: 81-6-6879-3449. E-mail: hayashi{at}moltx.med.osaka-u.ac.jp.

Journal of Virology, February 2000, p. 1241-1251, Vol. 74, No. 3
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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