Multiple liver-specific factors bind to the hepatitis B virus core/pregenomic promoter: trans-activation and repression by CCAAT/enhancer binding protein.

Abstract

The human hepatitis B virus (HBV) is a hepatotropic virus that replicates through an RNA intermediate referred to as the pregenome. The promoter that directs the synthesis of the pregenome and several other transcripts with heterogeneous 5' ends is of particular interest because ofits role in regulating key functions during the viral life cycle. We have examined the liver-specific characteristics of this promoter by DNA-protein interactions and by demonstrating the in vivo function of the promoter using the luciferase reporter gene expression system. The DNA-protein interactions in this region appear to be almost entirely liver-specific. Among these, a liver-specific nuclear factor, CCAAT/enhancer binding protein, binds to at least five sites on this promoter. Transient cotransfection experiments using CCAAT/enhancer binding protein expression vectors and the core promoter in the context of either the native hepatitis B virus genome or the luciferase reporter gene demonstrate that CCAAT/enhancer binding protein at low concentration modestly activates expression from the core promoter but represses at high concentration.The human hepatitis B virus (HBV) causes acute and chronic hepatitis, and the infection has been associated with hepatocellular carcinoma (1). There are four genes encoded by the viral genome: SipreS, Cle, Pol, and X (1). The transcription of these genes is controlled by at least four promoters (1-7). Additionally, an enhancer element has been identified in the HBV genome (8, 9). The core or pregenomic promoter regulates synthesis ofthe 3.6-kilobase (kb) RNAs that include mRNAs for several viral proteins (core, e, and pol) and the pregenomic RNA. Transcriptional regulation of these viral macromolecules makes this promoter a key element of the viral life cycle. The core promoter exhibits enhancerdependence for efficient liver-specific activity (6,(9)(10)(11)(12).In this study, we have used a series of restriction fragments and deletion mutations to define the boundaries of the HBV core/pregenomic promoter by using the firefly luciferase reporter gene (13). Furthermore, DNase I protection analysis was employed to identify nucleotide sequences that are binding sites for trans-acting cellular factors. This analysis revealed at least seven regions of DNA-protein interactions, most of which appear to be liver-specific. We show here that the CCAAT/enhancer binding protein (C/EBP), a liverspecific factor, binds to at least five sites in the promoter region. C/EBP was first purified from rat liver and shown to bind to several viral and cellular promoters/enhancers (14)(15)(16)(17)(18).To assess the role of C/EBP in the core promoter function, cotransfection experiments were carried out with a C/EBP expression vector and the core promoter linked to a reporter gene or in the context ofHBV genome. These studies indicate that C/EBP, at low concentrations, can function as a modest trans-activator of the core promoter. Increasing concentrations of C/EBP, however, resulted in trans-repression of the core ...

High Prevalence of 1762T 1764A Mutations in the Basic Core Promoter of Hepatitis B Virus Isolated From Black Africans With Hepatocellular Carcinoma Compared With Asymptomatic Carriers

“…Liver-specific nuclear factor CCAAT/enhancer binding protein bind to this region to activate transcription. 33,34 Recently, suppressed HBeAg synthesis has been shown to result from decreased binding of liver-enriched factor to the TA-rich region spanning nucleotides 1758-1764. 44,45 Although we cannot exclude the possibility that the differences in HBeAg titer were caused by host differences between ASCs and HCC patients rather than differences in the virus, we consider this to be unlikely.…”

Detection of silencer activity in the long control regions of human papillomavirus type 6 isolated from both benign and malignant lesions

“…Thus the context of the sequence may be important. Other putative cis elements within region 1 + 2 for which there are precedents for involvement in negative regulation in other systems include the C/EBP, AP1 and GRE sites (Hay et al, 1989;Lucibello et al, 1990;Lopez-Cabrera et al, 1990). An alternating purine-thymidine-rich sequence and/or short repeats located 5' to the late polyadenylation signal, at nt 7407 (Farr et al, 1991), may also play a role in negative regulation (Santoro et al, 1984;Yu & Manley, 1986).…”

LRH-1/hB1F and HNF1 synergistically up-regulate hepatitis B virus gene transcription and DNA replication

“…The liver-specific activity. of ENII is regulated by multiple liver-enriched transcription factors, including hB1F, HNF1, HNF3β, HNF4, and C/EBP [3][4][5][6][7][8][9]. The interaction bewteen each of these factors and ENII has been studied in great detail, but less is known on the interplay of these important regulators, which we think is crucial for the function of ENII as well as the life cycle of HBV.…”