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J. Biol. Chem., Vol. 276, Issue 26, 24059-24067, June 29, 2001
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From the Institute of Microbiology and Immunology, School of Life
Science, National Yang-Ming University, Shih-Pai,
Taipei 112, Taiwan, Republic of China
A negative regulatory element (NRE) is located
immediately upstream of the upstream regulatory sequence of core
promoter and second enhancer of human hepatitis B virus (HBV).
NRE represses the transcription activation function of the upstream
regulatory sequence of core promoter and the second enhancer. In this
study, we described the cloning and characterization of an NRE-binding protein (NREBP) through expression cloning. NREBP cDNA is 8266 nucleotides in size and encodes a protein of 2386 amino acids with a
predicted molecular mass of 262 kDa. Three previously described cDNAs, DBP-5, SONB, and SONA, are partial sequence and/or
alternatively spliced forms of NREBP. The genomic locus of the
NREBP/SON gene is composed of 13 exons and 12 introns. The
endogenous NREBP protein is localized in the nucleus of human hepatoma
HuH-7 cells. Antibody against NREBP protein can specifically block the
NRE binding activity present in fractionated nuclear extracts in gel
shifting assays, indicating that NREBP is the endogenous nuclear
protein that binds to NRE sequence. By polymerase chain
reaction-assisted binding site selection assay, we determined that the
consensus sequence for NREBP binding is GA(G/T)AN(C/G)(A/G)CC.
Overexpression of NREBP enhances the repression of the HBV core
promoter activity via NRE. Overexpression of NREBP can also repress the
transcription of HBV genes and the production of HBV virions in a
transient transfection system that mimics the viral infection in
vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AY026895. The nucleotide sequences reported in this paper have been
submitted to the DDBJ/GenBankTM/EBI Data Bank with
accession numbers AP000046 (human NREBP/SON gene), and EMBL with
accession numbers X63071 (human DBP-5 cDNA), X63751 (human SONB
cDNA), and X63753 (human SONA cDNA).
Transcription Repression of Human Hepatitis B
Virus Genes by Negative Regulatory Element-binding Protein/SON*
,,
*
This study was supported by National Science Council Grants
NSC-87-2315-B-010-002MH, NSC88-2315-B-010-012MH, and
NSC89-2315-B-010-007MH, National Health Research Institute Grants
NHRI-GT-EX89B906L and NHRI-EX90-8906BL, and Veterans General Hospital
(Republic of China) Grant V89-383-4.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The first two authors contributed equally to this work.
§
Recipient of an award from the Medical Research and Advancement
Foundation in memory of Dr. Chi-Shuen Tsou. To whom
correspondence should be addressed. Tel.: 886-2-28222400; Fax:
886-2-28212880; E-mail: lpting@ym.edu.tw.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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