Functional analysis of a liver-specific enhancer of the hepatitis B virus.

Abstract

The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liverspecific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAA-CA(GTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.The human hepatitis B virus (HBV) infects hepatocytes and causes acute and chronic hepatitis. Expression of the HBV genes appears to be modulated by transcriptional control elements that display liver specificity. This is facilitated by the interaction of trans-acting cellular factors with upstream regulatory sequences. There are four genes encoded by the viral genome (Fig. 1A): S/preS, C/e, Pol, and X (1), whose transcription is controlled by four promoters (see review, ref. 1). Two enhancer elements have been identified in the HBV genome by the use of reporter genes in heterologous systems (3-5), both of which display liver specificity (3-7). The enhAncer element, termed enhancer I, is located between 966 and 1308 nt (3, 4, 7) and also has been speculated to contain the functional domains of the X gene promoter. This enhancer has been previously shown to direct its influence on the promoters of C, X, and, to a modest extent, the S ORF (8-12). Deletion of this enhancer from the genome results in a general decline of transcription from vi-al promoters (13).Analysis of DNA-protein interactions of both enhancers has revealed binding regions for several cellular transcriptional factors (6, 14-17). These protein binding sites display homology to known sequence motifs for which factors have been identified and purified. Some of these interactions have been shown to be liver specific (6,(14)(15)(16)(17). A second element, designated as enhancer II, was identified relatively recently (5) and maps approximately within the core/pregenomic promoter at nt 1645-1803 in the HBV adw2 genome.In this report, using deletion mutagenesis studies of the enhancer/X promoter complex, we have identified key elements crucial for enhancer function and those required for X promoter activity. Two regions of DNA-protein interaction were shown to be essential for enhancer activity. The transcription factor EF-C binds to one of these sites (18), and the other site binds a...

RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins.

“…RFX1 is a component of the nuclear complexes that have been referred to independently as EF-C, EP, MDBP (37), or rpL30a (35). EF-C and EP are nuclear proteins binding to partially palindromic sequences called EF-C or EP sites present in the enhancers of several viruses including polyomavirus, HBV, and CMV (7,10,15,28,29,42). MDBP was first identified as a protein that binds preferentially to 5-methylcytosine-rich DNA and was subsequently shown to be a site-specific DNA-binding protein that binds to the same sites as EF-C but also binds to certain EF-C-related sequences (MDBP sites) in a manner that is strictly dependent on the presence of methylated CpG dinucleotides (10,18,22,45,(50)(51)(52).…”

“…RFX1 is a transcription factor that was initially cloned by virtue of its affinity for the X-box motif (31, 32), a conserved cis-acting regulatory element present in the promoters of all major histocompatibility complex (MHC) class II genes from all species examined (for reviews, see references 3, 13, and 41). RFX1 was subsequently also found to bind with high affinity to inverted repeats known as enhanced factor C (EF-C) sites (also called EP or methylation-dependent DNAbinding protein [MDBP] sites), which are cis-acting regulatory elements present in the enhancers of several unrelated viruses, including hepatitis B virus (HBV), polyomavirus, and cytomegalovirus (CMV) (7,10,15,28,29,42,(50)(51)(52). Among these EF-C sites, the functional importance of the site in the HBV enhancer (EnhI) has been most clearly demonstrated (7,11,15,29,37,42 .…”

The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors.

“…contains binding sites for liver-specific transcription factors such as C\EBP as well as for ubiquitous transcription factors like AP1, CREB, NF1 and a NF-κB-like motif (Trujillo et al, 1991 ;Gustin et al, 1993). To determine the factors mediating the transactivating effect, we tested which of the corresponding factor-binding sites are essential for transactivation by MHBs t"'( .…”

Hepatitis B virus infection and primary hepatocellular carcinoma

“…The important roles of the products of these genes in the regulation of cell growth and differentiation are consistent with the conclusion that their altered expression may be a factor in the etiology of some cases of PHC, but such integration events seem to occur in only a tiny fraction of human PHC patients examined. In this context, it is important to point out that the patterns of HBV expression and replication are dependent on the state of differentiation of an infected cell (5,45,148,246,282,369 (24,25) (Fig. 3) Putative role of other oncogenes.…”