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. Infection of hepatitis B virus (HBV)1 causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma. HBV is a small enveloped DNA virus with a partially double-stranded 3.2-kb genome. The genome contains four partially overlapping open reading frames (ORFs) coding for the surface, core, polymerase, and X proteins. The transcription of these open reading frames is under the control of four promoters (two surface promoters, one core promoter, and one X promoter) and two enhancers (enhancer I and enhancer II). Core promoter produces two 3.5-kb RNAs: the precore and pregenomic RNAs. Precore RNA encodes precore protein and e antigen. Pregenomic RNA not only serves as the mRNA that encodes core and polymerase proteins but also can be packaged into nucleocapsids along with viral polymerase, serving as the template for reverse transcription. Regulated expression of pregenomic RNA plays a pivotal role in the control of the viral replication cycle. The core promoter can be divided into two elements: the basal core promoter (BCP) and the core upstream regulatory sequence (CURS). CURS can activate the adjacent downstream BCP activity in cis. Interestingly, the CURS is also colocalized with the second enhancer (ENII) in the HBV genome (1). The ENII can activate the surface and X promoters in a position-and orientation-independent manner (2). The CURS/ENII displays a differentiated liver cell specificity (3), which is the combined effect of several liver-enriched transcription factors, such as ...