The TATA-less promoter of hepatitis B virus S gene contains a TBP binding site and an active initiator

Virus Research 49 (1997) 1 – 7

The TATA-less promoter of hepatitis B virus S gene contains a TBP binding site and an active initiator V. Bogomolski-Yahalom a, A. Klein b, I. Greenblat b,c, Y. Haviv d, R. Tur-Kaspa b,c,* a

Department of Medicine, Hadassah Uni6ersity Hospital, Mount-Scopus, Jerusalem b Li6er Unit, Hadassah Uni6ersity Hospital, Ein Kerem, Jerusalem c Di6ision of Medicine, Hadassah Uni6eristy Hospital, Ein Kerem, Jerusalem d Department of Molecular Genetics and Virology, Weizmann Institute of Science, Reho6ot, Israel Received 10 August 1996; accepted 14 November 1996

Abstract The surface antigen (S) gene promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of a 2.1 kb mRNA which encodes the preS2 and S polypeptides. The preS2/S promoter does not contain a classical TATA box, and transcription regulation of the preS2/S gene has not been fully elucidated. We analysed two regions involved in preS2/S gene transcription of the HBV adw subtype: the diverged TATA box and a putative initiator element. We demonstrated sequence specific promoter activity of the putative TATA-like sequences in the preS2/S gene promoter ( − 25 to −32 bp). Using end labeled synthetic oligonucleotides we observed specific binding of nuclear extracts to the diverged TATA sequence, that was significantly reduced using a mutated oligonucleotide. Specific binding of yeast TBP to the diverged TATA sequence was shown which was increased in the mutant containing a classical TATA box. We analysed the proposed initiator (Inr) sequence of the preS2/S promoter region ( −13 to −16 bp). Deletion of the Inr element markedly reduced promoter activity as assessed by CAT expression. Gel shift assays showed specific binding of nuclear extracts to wild type but not to mutant Inr. Expression studies with double mutants of the diverged TATA and the Inr element established that both elements are active in transcription regulation. © 1997 Elsevier Science B.V. Keywords: Hepatitis B virus; TATA-less promoter; Initiator; PreS2/S promoter

* Corresponding author. Department of Medicine D, and the Liver Institute, Rabin (Belinson) Medical Center, PetachTikva 49100, Israel.

The hepatitis B virus (HBV) genome contains different promoters: the core promoter, preS1 and preS2/S promoter and the X gene promoter. The preS1 promoter directs the synthesis of the large envelope protein. The surface antigen (S) gene promoter directs the synthesis of a 2.1 kb mRNA

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which encodes the middle (preS2) and major (S) polypeptides (Ganem and Varmus, 1987). Eukaryotic protein coding genes can be classified as containing or lacking a TATA box (TATAAA) which is usually located 25 – 30 bp upstream from the transcriptional start site (Pugh and Tijan, 1991). The TATA box binds TFIID, other transcription factors (TFIIA, TFIIB, TFIIF, TFIIE, TFIIH) various transcription associated factors known as TAF’s and RNA polymerase II (Buratowski, 1994). In some promoters which lack a TATA box, initiator elements were identified (Weis and Reinberg, 1992) with a loose consensus sequence (Py Py A+1N T/A Py Py) (Javahery et al., 1994). Initiator elements usually overlap the transcription start site and are recognised by TFIID (Kaufmann and Smale, 1994). Initiators enhance promoter strength either in TATA-less or in TATA containing promoters (Weis and Reinberg, 1992; Kaufmann and Smale, 1994). During natural infection with HBV, 98% of the transcripts initiate from the PreS2/S promoter (Siddiqui, 1991), but unlike the PreS1 promoter, the PreS2/S promoter does not contain a classical TATA box (De-Medina et al., 1988). Sequence analysis suggested the presence of a diverged TATA box (TAAGAGA) from −23 to − 17 in the adw and ady subtypes. Deletion analysis in adw has revealed the region −40 to − 9 to be crucial to promoter activity (De-Medina et al., 1988). The presence of a putative initiator element in this region (− 16 to − 13) raises the possibility that the PreS2/S promoter in the adw type has at least two transcription control elememts. We have analyzed those two regions. To assess the significance of the diverged TATA box in the preS2/S promoter, we used plasmid p16II constructed by cloning the S promoter region (−48 to +30) of the adw subtype into the BamHI site and the chloramphenicol acetyl transferase (CAT) gene sequences to the HindIII site of plasmid pSp64 (Melton et al., 1984) (Fig. 1). Plasmid p16E was constructed by cloning the SV-40 enhancer region at the EcorV site of plasmid p16II (Fig. 1). SK-Hep 1 (poorly differentiated human hepatoma) cells (ATCC, 1994) were transfected with

10 mg plasmid DNA per 10 cm plate, using the calcium phosphate coprecipitation method (Graham and Van der Eb, 1973). Extracts were prepared 40 h after transfection and CAT activity measured according to the method of Gorman et al. (1982). Low levels of CAT activity in the p16II plasmid which were increased in the presence of the enhancer element (p16IIE) were obtained (data not shown). In order to analyze the role of the diverged TATA box sequence (− 32 to − 25 bp), we constructed mutants either closely resembling a classical TATA box (TA rich-p16E T) or GC-rich (less TATA like -P16ELT) (Fig. 1) and

Fig. 1. Construction of plasmids and mutants. (A) Plasmid P16II was constructed by cloning the S promoter region (−48 to + 35 bp) of the adw subtype into the BamHI site of pSp64 (9) and the chloramphenicol acetyl transferase (CAT) gene sequences from pSV0CAT into the HindIII site. Plasmid p16E was constructed by cloning the SV-40 enhancer region at the EcoRV site of plasmid p16II. (B) Mutant plasmids — p16E T was constructed using an oligomer corresponding to the − 48 to +35 of HBV adw genome except for a 4 bp change from AGAG to TATA from − 28 to −24 bp. The mutation was introduced by excision of a 38 bp fragment between MstII – SacI and exchanging it with a synthetic oligomer. The oligomer was synthesized by the Molecular Biology Unit, Hadassah University Hospital. p16E lT was constructed using an oligomer corresponding to bp − 48 to +35 of the HBV adw genome except for a 5 bp change from AGAG to CCGGA from −29 to −24 bp. The mutation was introduced by excision of a 38 bp fragment between MstII and SacI and exchanging it with a synthetic oligomer.

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analyzed the mutants for promoter activity and the ability to bind transcription factors. CAT expression obtained by the mutated GC-rich plasmid was at least 3 fold lower than that seen with the wild type sequence (data not shown). These results indicate that the diverged TATA sequences might have significant promoter activity. Binding of nuclear extracts and TATA binding protein to synthetic oligonucleotides corresponding to the diverged TATA box, TA rich mutant and GC rich mutant was determined. Nuclear extracts of SK Hep 1 cells were prepared according to Schreiber et al. (1989). Binding of nuclear extract to the labeled mutated oligomers was preformed and analyzed by gel shift assay, using unlabeled oligomers to show binding specificity (ratio labeled to unlabeled oligonucleotides = 1:500), (Fig. 2). Specific binding of nuclear extract to the wild type diverged TATA box was evident. Mutation of the diverged TATA region to a GCrich sequence significantly diminished binding of nuclear extracts. It is noteworthy that in the mutated TA-rich sequence, binding to nuclear factors was observed (Fig. 2). Binding assays with yeast recombinant-TATAbinding-protein (yTBP) (Pugh and Tijan, 1991; Kao et al., 1990; Hoffman et al., 1990) and oligomers corresponding to the wild type sequence, or TATA and CCGGA mutants were preformed. Mobility shift assays revealed that yTBP binds to the wild type sequence (Fig. 3). Mutation of the wild type sequence to a GC-rich sequence abolished yTBP binding. It is of note that a classical TATA sequence binds yTBP. Analysis of the sequences in the vicinity of the diverged TATA sequence revealed a presumable initiator (Inr) element: −16 to −13 bp (CTCA). As initiator elements are known to play a role in both TATA-less or TATA-containing promoters, we were interested to determine the relative significance of the Inr in the HBV preS2/S promoter which contains a diverged TATA box. For that purpose, 32P-end labeled oligomers spanning this region and a mutated sequence (deletion of the sequence −15 TCA −13), were synthesized. Gel shift assay binding of SK HEP 1 nuclear extract to the oligomers show (Fig. 4) that an oligomer corresponding to the initiator sequence − 16 to

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Fig. 2. Binding of SK-HEP1 nuclear extract to preS2/S promoter. Binding was performed with oligonucleotides that were end-labeled with 32P at the 3% end. Reaction assay contained 5000 cpm of labeled dS oligomer, 10 mM Hepes pH 7.9, 4 mM MgCl2, 5 mM (NH4)2 SO4, 8% glycerol, 2% PEG, 50 mM KCl, 1 mM b67C:DOCSVRSVOL49.1 -mercaptoethanol, 1 mg poly dIdC or dGdC, 0.1 mM EDTA and nuclear extracts, incubated at 25°C for 30 min and assayed on a 5% acrylamide gel. Nuclear extracts from SK-HEP 1 cells were prepared (Gorman et al., 1982) and incubated with radioactive synthetic oligomers corresponding to the wild type HBV adw subtype promoter region from − 48 to +35 bp (lane 1). In lane 3, binding of oligomers corresponding to the TA rich mutant is shown. Binding was diminished with oligomers corresponding to the GC rich mutant (lane 2). In lanes 1a, 2a and 3a unlabelled oligonucleotides were added to establish binding specificity, (ratio labeled to unlabeled oligonucleotides = 1:500). Arrows indicate the shifted bands.

− 13 bp (CTCA) binds SK-HEP 1 nuclear extracts. On the other hand, deletion of the Inr sequence abolished nuclear extract binding. In order to evaluate the significance of the Inr to the promoter activity we created a plasmid

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Fig. 3. Binding of yeast TBP to the diverged TATA sequence of the preS2/S promoter. Cloned yTBP was over-expressed in bacteria and purified using standard procedures (Kao et al., 1990; Hoffman et al., 1990; Pugh and Tijan, 1991). Mobility shift assays were performed as described in Fig. 2. In lane 1 binding of yTBP to the preS2/S promoter of wild type HBV adw (diverged TATA) is shown. Mutation of the wild type sequence to a GC-rich sequence (as in plasmid p16IIE LT) abolished yTBP binding (lane 3). A classical TATA sequence (as in plasmid p16IIE T) binds yTBP (lane 2). Binding specificity is shown by addition of unlabelled oligonucleotides (lanes 1a, 2a, 3a), (ratio labeled to unlabeled oligonucleotides= 1:500).

mutant with deletion of the Inr sequence. We digested plasmid p16IIE with Bsu36I (New England Biolabs, USA) and Mung Bean endonuclease (Boehringer Monnheim, GMBH Germany), the plasmid was religated with T4 ligase (Boehringer Monnheim, GMBH Germany). Sequencing the mutant demonstrated that 3 bp (i.e, − 15 TCA − 13) were deleted. The plasmid was designated p16EV. SK HEP 1 cells were transfected with p16IIE and p16EV as previously described. CAT assay performed according to Neuman et al. (1987) showed a marked reduction of CAT activity induced by the mutated plasmid p16EV (Fig. 5). The relative significance of the diverged TATA box and the Inr to promoter activity, was studied by mutating the Inr sequence in the plasmids p16IIE T and p16IIE LT (Fig. 5). To create the mutations plasmids p16IIE T and p16IIE LT were digested with Bsu36I, Mung Bean end nuclease was applied and the plasmids were religated with

T4 ligase as previously described. Plasmids were transfected to SK HEP 1 cells and CAT activity determined. Fig. 5 shows the relative CAT activities induced by the various CAT plasmids. Mutation of the diverged TATA sequence into a GC-rich (less TATA-like) sequence containing the wild type Inr sequence resulted in reduction of CAT activity by 4-fold. Moreover, deleting the Inr sequence reduced CAT expression to only 8% of the CAT actvity induced by the wild type sequence. Interestingly, deletion of the Inr sequence in the plasmid with the wild type sequence reduced CAT expression by more than 10-fold. Mutating the diverged TATA to a TA-rich (TATA-like sequence) resulted in  2 fold reduction as compared with the wild type sequence. In this plasmid deletion of the Inr sequence further reduced CAT activity. These results show that both the Inr and the diverged TATA box function as active promoter elements in the preS2/S promoter. The initiator element CTCA plays a criti-

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cal role in the preS2/S promoter in the absence of a classical TATA sequence. Its effect on promoter activity as measured by CAT expression is less evident when the diverged TATA sequence was replaced with a classical TATA sequence. In the ayw subtype a diverged TATA box is not evident (-35 CCTTTGAAGAA-25). Deletion Fig. 5. CAT activities of preS2/S promoter mutants. Determination of the relative CAT enzyme activity (Neuman et al., 1987) in cellular extracts obtained from SK-HEP1 cells after transfection (Graham and Van der Eb, 1973) of the reporter plasmids (10 mg per 10 cm plate). p16IIE contains the wild type sequence of the HBV adw preS2/S promoter (−48 to +35 bp) with both the diverged TATA sequence and the initiator. p16IIE T and p16IIE LT contain the Inr sequence but the diverged TATA sequences were mutated to either TA-rich (p16IIE T) or GC-rich (p16IIE LT). In the plasmids p16IIEV, p16IIEV T, p16IIEV LT the Inr sequence was deleted from − 15 to − 13 bp (CTC). p16IIEV has the wild type diverged TATA sequence, p16IIEV LT contains the GC-rich mutation and p16IIEV T the TA-rich mutation. CAT activity is expressed as the relative activity in relation to the CAT activity induced by p16IIE.

Fig. 4. Nuclear extract binding to the Iniator and mutated Initiator sequence. Oligomers corresponding to preS2/S promoter region ( − 48 to + 35 bp) of the HBV adw subtype and the mutated Inr (deletion of −16 to − 13 bp, CTC) were synthesized by the Molecular Biology Unit, Hadassah University Hospital. Nuclear extracts from SK-HEP 1 cells were prepared (Schreiber et al., 1989). Binding was performed as described in Fig. 2. Binding of SK-HEP 1 nuclear extracts to an oligomer with the Inr sequence is demonstrated in lane 1. There was no binding of SK-HEP 1 nuclear extract to an oligomer from which the Inr was deleted (lanes 2, 2a). Lanes 1a and 2a show the effect of adding unlabelled oligonucleotides (ratio labeled to unlabeled oligonucleotides= 1:500).

analysis (Raney et al., 1991) demonstrated more than a 10-fold reduction in relative luciferase expression when the sequences between − 44 and −23 were deleted. It should be noted that deletion of bp − 22 to + 16 which contain the presumable initiator element reduced luciferase activity to 0.71 and 0.55 compared with the wild type sequence in HepG2.1 and HuH7 cells respectively. The diverged TATA sequence, analyzed in this study is found in the genome of the adw and ady but not in the ayw subtypes (Shaul, 1991), although both subtypes were shown to be infective in vivo (Tabor et al., 1983). We were able to demonstrate that this sequence plays a role (although less than that of the Inr sequence) in the transcription of the preS2/S promoter of the adw subtype. Our results are somewhat different from the work of Zhou and Yen (1991). Using deletion analysis, they argue that the divergent TATA box has a minor contribution to the preS2/S promoter activity. We have found that the divergent TATA box cooperates with the initiator, but that the initiator has a more prominent role than that of the divergent TATA box.

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Four functional binding sites for the Sp1 were found in the regulatory regions of the major surface antigen promoter and are thought to contribute to the level of expression from this promoter during natural infection (Raney et al., 1992). Colgan and Manley (1995) demonstrated that the complex of an initiator with Sp1 binding sites in a TATA less promoter is more potent than a promoter with a TATA box. These findings might explain the fact that during natural infection 98% of transcipts initiate from the preS2/S promoter, even though it lacks a TATA box. The minimal promoter of the precore and pregenomic RNA of the HBV contains overlapping sequences that can function both as an initiator and as a TATA element. These two elements are required for efficient transcription as in the preS2/S promoter. Our results suggest that the preS2/S promoter in the HBV adw subtype contains two control elements; a relatively weak diverged TATA element and an initiator element which is the main factor determining transcription in the preS2/S promoter.

Acknowledgements We are indebted to Dr Deborah Rund for critical review of the article. This work was supported in part by the Binational Science Foundation USA—Israel (to RTK), the Joseph Birenbaum Grant (to RTK) and by the Joint Fund Hadassah-Hebrew University (to VBY).

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