JOURNAL OF VIROLOGY, Mar. 1994, P. 1758-1764 Vol. 68, No. 3 0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology Repression of Enhancer II Activity by a Negative Regulatory Element in the Hepatitis B Virus Genome WEI-YU LO AND LING-PAI TING* Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Shih-Pai, Taipei 11221, Taiwan, Republic of China Received 13 September 1993/Accepted 29 November 1993 Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation- dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined efforts of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression. Hepatitis B virus (HBV) infection is one of the world's the upstream stimulatory function of the CURS on the BCP. In leading health problems. Chronic carriers of HBV have a addition, the NRE can dominantly repress the enhancer greatly elevated risk of developing cirrhosis and hepatocellular activity of enhancer II in differentiated liver cells. This latter carcinoma (1, 21). HBV is one of the smallest DNA viruses; it repressive function appears to be dependent on enhancer II in has a partially double-stranded 3.2-kb viral genome. There are that the repressive effect is seen mainly in the presence of an four open reading frames, which code for the surface, core, intact enhancer II. Fine mapping by linker-scanning analysis polymerase, and X protein (2, 5, 22). The transcription of these reveals that the sequence between nt 1616 to 1621 is required genes is under the control of four promoters, two for surface for the repressive effect on enhancer II. (SPI and SPII), one for core and polymerase (CP), and the other for X (XP) (3, 4, 8, 16, 24, 29, 30). Two enhancers, enhancer I and enhancer II, have been identified so far in the MATERIALS AND METHODS HBV genome. These enhancers have been shown to stimulate Plasmid construction and preparation. The HBV sequence the viral promoters in a hepatocyte-specific manner, which may used in this study is of the adw subtype. Numbering of the explain in part the hepatotropism of this virus (8, 20, 23, 26, HBV sequence begins at the unique EcoRI site, which is nt 1. 27). All reporter plasmids used in transfection experiments contain We have previously reported the characterization of en- a head-to-tail trimeric tandem repeat, referred to as A3, of a hancer II. Located downstream of enhancer I and within the X 237-bp BclI-BamHI fragment from the simian virus 40 (SV40) open reading frame, this enhancer is composed of two inter- polyadenylation signal. A3 is placed 5' of sequences assayed acting sequence motifs, a 23-bp box a (nucleotides [nt] 1646 to for promoter activity and has been shown to stop transcription 1668) and a 12-bp box a (nt 1704 to 1715). Cooperation of the readthrough from spurious upstream initiation. The parental two elements is required for enhancer function. This enhancer plasmid containing A3 on a pGEM backbone is designated as can stimulate the transcriptional activities of SPI, SPII, and XP pA3/RIdB. in a position- and orientation-independent manner (25-27). pHBV3.8 was constructed by three-way ligation. The EcoRI- Interestingly, both box a and box a are also constituents of FspI fragment containing the A3 and core promoter sequence the core upstream regulatory sequence (CURS) and can (nt 1402 to 1804) was taken from pA3(1402-1851)CAT. This positively regulate the transcription of the downstream basal fragment was ligated to the FspI-PstI fragment from HBV (nt core promoter (BCP) individually. Box a and box (3 can 1805 to 25) and an EcoRI-PstI-restricted pGEM vector back- strongly and moderately, respectively, stimulate the activity of bone. The resulting plasmid was further extended with the BCP in only a position- and orientation-dependent fashion PstI-fragment containing the HBV sequence from nt 25 to (24). Although the trans-acting factor(s) that mediates these 1990 isolated from pSpHBsl775 at the unique PstI site to functions is not yet fully understood, C/EBP-like protein(s) create pHBV3.8. and HNF-4 have been shown to be likely candidates (9, 16, 26). Plasmids pHBV3.65, pA3(1402-1851)CAT, pA3(1636- In this paper we report the identification of a negative 1851)CAT, and pA3(1744-1851)CAT were described previ- regulatory element (NRE) upstream of box ox that abolishes ously (24). Plasmid pA3(1613-1851)CAT was constructed by replacing the sequence from nt 1402 to 1851 of pA3(1402- 1613 to * Corresponding author. Mailing address: Graduate Institute of 1851)CAT with the 239-bp NlaII-RsaI fragment (nt Microbiology and Immunology, National Yang-Ming Medical College, 1851) of the HBV sequence. Shih-Pai, Taipei 11221, Taiwan. Phone: 886-2-8222400. Fax: 886-2- Plasmid pA3SVpCAT contains the bacterial chlorampheni- 8212880. col acetyltransferase (CAT) gene driven by the SV40 early 1758 VOL. 68, 1994 REPRESSION OF HBV ENHANCER 11 1759 promoter. The 129-bp Pstl fragment containing the HBV Assay for endogenous DNA polymerase activity. To assay for sequence from nt 1613 to 1741, derived from p(1613-1741)/ endogenous DNA polymerase activity, the culture supernatant 3Zf(+), was subcloned into the PstI site immediately down- was collected 3 days after transient transfection, treated with stream of the polyadenylation site in pA3SVpCAT. The same 1% Nonidet P-40 for1 h at room temperature, and centrifuged 129-bp BamHI fragment eluted from p(1613-1741)/3Zf(+) at 17,000 x g for 30 min at 4°C. The supernatant was collected was inserted into the BamHl site upstream to the promoter and recentrifuged at 225,000 x g for 1 h at 4°C. The pellet in pA3SVpCAT. The resulting plasmids which carry insertions from the second centrifugation, which contains HBV viral core at downstream or upstream positions in either orientation particles, was resuspended in TNE buffer (10 mM Tris-HCl were obtained and designated as pA3SVpCAT(1613-1741), [pH 7.5], 50 mM NaCl, 0.1 mM EDTA) and assayed for pA3SVpCAT(1741-1613), pA3(1613-1741)SVpCAT, and endogenous polymerase activity as previously described (24). pA3(1741-1613)SVpCAT, respectively. The set of plasmids Preparation and heparin-Sepharose fractionation of nu- shown in Fig. 4 was constructed by insertion of the synthetic clear extracts. Nuclear extracts from the differentiated human oligonucleotides hepatoma cell line HepG2 were prepared as previously de- 1613 1636 scribed (3). The extracts were fractionated at 4°C as previously 0 described (26). The crude and fractionation nuclear extracts 5'-gatctGAGACCACCCTGAACGCCCATCAGg-3' were aliquoted, quickly frozen under liquid nitrogen, and kept 3'-aCTCTGGTGGCACTTGCGGGTAGTCcctag-5' frozen at - 70°C. (coding strand on top; the BamHI-BglII linker sequence is Gel shift analysis. The probe was prepared with annealed shown in lowercase letters) into the BamHI site located double-stranded oligonucleotides (200 ng) corresponding to upstream of the promoter or downstream of the polyadenyla- the NRE sequence of HBV (as shown in plasmid construc- tion site of pA3SVpCAT, respectively. tions) and end labeled via fill-in with [ax-32P]dATP (3,000 The CAT fragments of pA3(1613-1636)SVpCAT and Ci/mmol; Amersham Corp., Amersham, England) at either pA3(1636-1613)SVpCAT were replaced with the CAT(1636- terminus of a BglII-BamHI linker site. 1741) fragments to generate pA3(1613-1636)SVpCAT(1636- Nuclear extracts (10 VLg) were incubated in a 20-,ul reaction 1741) and pA3(1636-1613)SVpCAT(1636-1741) shown in Fig. mixture containing 1 ,ug of poly(dI-dC)(dI-dC) (Pharmacia, 7. Inc.), IOx binding buffer (170 mM N-2-hydroxyethylpipera- The synthetic oligonucleotides corresponding to the wild- zine-N'-2-ethanesulfonic acid HEPES [pH 7.9], 20% glycerol, type and mutant sequences of HBV from 1613 to 1668 (see 4.2 mM EDTA, 3 mM dithiothreitol, 100 mM KCl, 62.5 mM Fig. 6) were annealed and inserted into the BamHI site MgCl2), 12.5% glycerol, and 2.2 x l10 cpm of labeled DNA. downstream of pA3SVpCAT to generate pA3SVpCAT(1613- Reaction mixture were incubated at 30°C for 30 min, and 1668WT), pA3SVpCAT(1613-1668AB), or pA3SVpCAT protein-DNA complexes were resolved on 4% polyacrylamide (1613-1668MT). The 1.9-kb SacI-XbaI fragments from plas- (acrylamide/bisacrylamide weight ratio, 30:1) made in 1 x TBE mids SVpCAT(1613-1668WT), SVpCAT(1613-1668AB), and (90 mM Tris-borate [pH 8.0], 2 mM EDTA). Electrophoresis SVpCAT(1613-1668MT) were first blunt ended with Klenow was performed at 150 V for 2.5 h at 4°C. The gel was dried and fill-in and used to replace the SacI-StuI segment in autoradiographed.