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

Abstract

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.Hepatitis B virus (HBV) causes acute and chronic hepatitis in humans, and chronic infection is closely associated with the development of hepatocellular carcinoma (3). HBV has a partially double-stranded 3.2-kb DNA genome containing four overlapping open reading frames (ORFs) encoding surface antigen (HBsAg), core/e antigen (HBc/eAg), polymerase, and X protein (19,47). Four promoters (CP, SPI, SPII, and XP) have been identified as cis regulators for transcription of the 3.5-, 2.4-, 2.1-, and 0.8-kb mRNAs, respectively. The 3.5-kb mRNA encodes viral polymerase and HBc/eAg and functions as the pregenome RNA for reverse transcription of the HBV genome as well (8,45,54,56). The 2.4-and 2.1-kb mRNAs are the templates for large and middle/major surface antigens, respectively (7,36,41,43), and the 0.8-kb mRNA is specific for X protein (42, 49). So far, two regions in the HBV genome have been shown to act as transcriptional enhancers. Enhancer I (EnI), which is located upstream of the X gene, displays a preference for hepatocytes (40, 48), while enhancer II (EnII), located just upstream of CP, shows highly restricted hepatocyte-specific activity (53, 57, 60); both enhancers stimulate transcription from the promoters (2,22,28,44,51,57,60). In transfection analysis with cloned HBV DNA, the 3.5-kb mRNAs were detected in well-differentiated human hepatoma cell lines but not in nonhepatic cells (9,46,50,55). This suggests that hepatocyte-specific factors are necessary for the transcription of pregenome RNA and that the hepatotropism of HBV replication might be attributable to the EnII function.EnII stimulates the transcript...

The Hepatitis B Virus Core Promoter Is Strongly Activated by the Liver Nuclear Receptor Fetoprotein Transcription Factor or by Ectopically Expressed Steroidogenic Factor 1

“…These experiments, however, used an HBV reporter construct from which FTF#1 was deleted and also used HeLa cells, in which transfected FTF may not make efficient use of its lower-affinity site (HNF4#2). Independent hFTF expression cloning was also achieved by yeast one-hybrid screening with the HBV segment including nts 1640 to 1663 (carrying the FTF#1 motif), and transfection assays conducted with hepatoma cells were also consistent with activation of the nt 1640 to 1663 segment by FTF (18).…”

HBV integrants of hepatocellular carcinoma cell lines contain an active enhancer

“…Previously, it was shown that a protein of about 50 Kd that was named PBF interacts with this cis element sequence (Kosovsky and Siddiqui, 1999). Given the fact that FTF binds and activates enhancer-II (Li et al, 1998, Ishida et al, 2000, Gilbert et al, 2000, the role of this protein in HBV life cycle becomes signi®cant. FTF is an orphan nuclear receptor of the Drosophila fushi tarazu F1 (FTZ-F1) family.…”

Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene

“…Luciferase constructs driven by Enh1 (PENS-Luc) or control (PS-Luc), pFPV3Luc, pFPV94Luc and p9SLuc (Kosovsky et al, 1996) were gifts from Aleem Siddiqui (University of Colorado Health Sciences Center, USA). Enh2 plasmid constructs PEII.1-Luc, PEII.2-Luc and TATA-Luc were gifts from Hisashi Ishida (Osaka University, Japan) (Ishida et al, 2000). pSpLuc, SpLuc, CpLuc and XpLuc were obtained from Alan McLachlan (The Scripps Research Institute, USA) (Raney et al, 1990).…”