A novel negative cis-regulatory element on the hepatitis B virus S-(+)-strand

Markus Wagner b,1, Michael Alt1, Peter Hans Hofschneider1 and Matthias Renner c,1

Department of Virus Research, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried , Germany1

Author for correspondence: Peter Hans Hofschneider.Fax +49 89 8578 2292. e-mail hofschneider{at}biochem.mpg.de


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis B virus (HBV) has a double-stranded DNA genome. The minus-strand contains coding regions for all known HBV proteins and most of the cis-regulatory elements. Little is known about transcription from the S-(+)-strand and its regulation. Thus, the presence of regulatory elements located on the S-(+)-strand was investigated by inserting nt 1038–1783 of HBV in both orientations between the human cytomegalovirus (HCMV) promoter and a luciferase gene. Transfection experiments revealed that the plasmid containing this HBV DNA fragment in an orientation allowing expression from the S-(+)-strand (antisense) led to inhibition of luciferase gene expression compared to the plasmid containing this sequence in an orientation that allows gene expression from the L-(-)-strand (sense). Deletion analyses delimit the sequence essential for the inhibitory effect to a 150 bp region that also carries part of the enhancerII/core promoter complex. However, the possible influence of this regulatory element has been excluded in various experiments. The repressing HBV sequence acts in an orientation- and position-dependent manner; no inhibition was observed when this DNA element was inserted upstream of the HCMV promoter or downstream of the luciferase gene. Northern blot analyses revealed reduced luciferase mRNA steady-state levels in cells transfected with constructs containing the essential HBV sequence in antisense orientation compared to plasmids containing this sequence in sense orientation. Since nuclear run-on experiments showed similar transcription initiation rates with these plasmids, the diminished luciferase mRNA steady-state levels must be due to altered stabilities, suggesting that nt 1783–1638 of HBV encode an RNA-destabilizing element.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Human hepatitis B virus (HBV) is a small enveloped virus with a partially double-stranded DNA genome of approximately 3·2 kb. Five open reading frames (ORFs) are located within the L-(-)-strand (minus-strand) representing the complete DNA strand. Four of these coding regions have been assigned to known viral proteins. The P gene encodes the viral polymerase, the preS/S region encodes the surface proteins LHBs, MHBs and SHBs, the C gene encodes the viral core protein and the X gene encodes the HBx transactivator (Ganem & Varmus, 1987 ). Until now, no protein product has been detected for ORF5, which lies within the X gene (Kaneko & Miller, 1988 ). Transcription of these genes is controlled by two enhancers and at least four promoters. The viral S-(+)-strand (plus-strand) contains only one ORF, named ORF6 (Miller, 1990 ; Miller & Robinson, 1986 ).

Two transcripts are derived from the HBV plus-strand. A 2·8 kb transcript initiates at nt 1861, 50 bp downstream of a TATA-like sequence (nt 1917–1921) which could serve as a promoter, and terminates at nt 2381 (Zelent et al., 1987 ). A second transcript of 0·7 kb in length starts at position 1634/1635 and terminates between nt 954 and 957 (Standring et al., 1983 ). These two transcripts contain the ORF6 region. However, no ORF6-specific protein has been shown in cell culture experiments yet. Velhagen et al. (1995 ) described a promoter activity between nt 1885 and 1575 which possibly could regulate transcription of the 0·7 kb RNA molecule. Recently, Shimoda et al. (1998 ) revealed direct evidence for the presence of an antisense promoter located in the ORF6 region of the woodchuck hepatitis virus (WHV).

Recently, beside the enhancerII/core promoter region (Yee, 1989 ; Yuh & Ting, 1990 ), several cis-regulatory elements have been detected within the ORF6 region of the plus-strand and the respective minus-strand region carrying the X ORF. However, none of these elements has been shown to be involved in ORF6 expression. Huang & Liang (1993 ) described a cis-activating element between nt 1236 and 1641 which facilitates nuclear export of HBV RNA by inhibiting splicing of these transcripts. This element consists of two subelements which function synergistically in an orientation- and position-dependent manner (Donello et al., 1996 ). As described by Donello et al. (1998 ), a similar post-transcriptional regulatory element is present in WHV indicating its conserved status among mammalian hepadnaviruses. In addition, a related element between nt 1238 and 1804 promotes HBV S-mRNA export of unspliced transcripts from the nucleus into the cytoplasm (Huang & Yen, 1994 , 1995 ). The binding of two cellular proteins seems to mediate this RNA transport (Huang et al., 1996 ).

Additionally, several negative regulatory elements have been identified in the investigated HBV sequence area. Guo et al. (1993 ) as well asChen & Ou (1995 ) characterized a DNA element which suppresses the core promoter by antagonizing the activity of the transcription factor HFN-4. The activity of this element is both cell type-dependent – it seems to act only in non-liver cells – and orientation-independent. A further negative regulatory element inhibits enhancerII activity, most likely mediated by masking enhancerII binding sites (Lo & Ting, 1994 ). This element is functional in an orientation- and position-dependent manner with respect to the enhancerII, but is orientation- and position-independent with respect to a test promoter (Lo & Ting, 1994 ).

As mentioned briefly, all of these elements have been shown to act in a minus-strand orientation-dependent manner or an orientation- independent manner and seem to be involved in regulation of minus- strand expression.

We report the presence of a novel cis-regulatory element which inhibits gene expression within the ORF6 region of the plus- strand in an orientation- and position-dependent manner. This element is located between nt 1638 and 1783 of the HBV genome. Northern blot experiments and nuclear transcription run-on analysis revealed that modulation of gene expression is based on different mRNA steady-state levels caused by altered mRNA stabilities suggesting that we have identified a new RNA-destabilizing element within the HBV genome.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Plasmid construction.
HBV fragments used for the determination of the minimal inhibiting HBV DNA sequence were derived from plasmid pX1-136 (Renner et al. , 1995 ), which contains nt 827–1783 of the HBV sequence, subtype ayw, followed by a SpeI-linker with stop codons in all three reading frames (New England Biolabs). Plasmid pX1-136 was digested with either SspI/SpeI, BamHI/SpeI, NcoI/SpeI or Dra I/SpeI to generate HBV fragments of nt 1638–1783, 1402–1783, 1374–1783 and 1038–1783, respectively. Cleavage of pX1-136 with SphI/ AvaI or AvaI/HincII resulted in HBV fragments of nt 1238–1465 and 1465–1686, respectively. After blunt-end formation with Klenow polymerase, the HBV fragments were inserted in either of the two orientations into plasmid pCMV- NCRluc linearized with HindIII (Alt et al., 1995 ), resulting in plasmids pHBV1638–1783s, pHBV1783–1638as, pHBV1402–1783s, pHBV1783–1402as, pHBV1374–1783s, pHBV1783–1374as, pHBV1038–1783s, pHBV1783–1038as, pHBV1238–1465s, pHBV1465–1238as, pHBV1465–1686s and pHBV1686–1465as. Control plasmid pCMV206NCRluc, which contains a 206 bp DNA sequence of pUC19 (Yannish-Perron et al., 1985 ) instead of a corresponding HBV fragment, was obtained by inserting a 206 bp HphI-digested fragment of pUC19 into plasmid pCMV-NCRluc, linearized with HindIII.

Plasmid pCMVluc was derived by inserting the 1904 bp Sal I fragment of pMamneoluc (Invitrogen) into the HindIII site of pRc/CMV (Invitrogen). Plasmids pHBV1638–1783CMVluc and pHBV1783–1638CMVluc, which contain the HBV DNA element from nt 1638 to 1783 upstream of the cytomegalovirus (CMV) early promoter/enhancer complex, were constructed by ligating the 145 bp SspI/SpeI fragment of pX1-136 with NruI- digested pCMVluc. Plasmid p206CMVluc, serving as a control vector, was derived by connecting a 206 bp HphI fragment of pUC19 with pCMVluc, linearized by NruI.

Plasmids pCMVlucHBV1638–1783 and pCMVlucHBV1783–1638, which contain the inhibiting HBV DNA element from nt 1638 to 1783 downstream of the luciferase gene were constructed by ligating the 145 bp SspI/SpeI fragment of pX1-136 with Nru I-digested pCMVluc. Plasmid pCMVluc206 was derived by ligating a 206 bp HphI-fragment of pUC19 with pCMVluc, linearized by ApaI.

To test the inhibitory effect of the HBV element in the context of a different promoter, plasmids pSVHBV1638–1783luc and pSVHBV1783–1638luc were used. Both constructs were generated by digesting pSV-NCRluc with HindIII, followed by blunt-end formation with Klenow enzyme and ligation with the 145 bp Ssp I/SpeI fragment of pX1-136. Plasmid pSV-NCRluc was derived by connecting the large HindIII/SalI fragment of pCMV-NCRluc with the small HindIII/SalI fragment of pSVßGal (Promega).

To generate plasmid p({Delta}CMV)HBV1638–1783, plasmid pCMV- NCRluc was digested with MluI and HindIII, to remove the CMV promoter region, and ligated to the 145 bp SspI/ SpeI fragment of pX1-136. Religation of the large Mlu I/HindIII fragment of pCMV-NCRluc resulted in plasmid p({Delta}CMV)NCRluc.

All of these plasmids were sequenced to confirm their structural integrity.

{blacksquare} Cell culture and transfection.
Tissue culture cells were grown at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum and 0·6% (w/v) penicillin/1·3% (w/v) streptomycin under an atmosphere of 93% air/7% CO2. Twenty- four hours prior to transfection, 1x106 HepG2 cells (ATCC HB8065), 5x105 Chang liver cells (ATCC CCL13) and 5x105 HeLa cells (ATCC CCL2) were seeded onto 6 cm tissue culture dishes. One microgram recombinant plasmid was introduced into the cells by the lipofection technique as recommended by the supplier (Life Technologies). Six hours after transfection, the medium was removed and cells were given fresh culture medium and incubated for 40 h.

{blacksquare} Luciferase assay.
Transfected cells were washed three times with PBS and lysed with 500 µl luciferase lysis buffer (25 mM Tris/H3PO 4, pH 7·8, 2 mM CDTA, 2 mM DTT, 10% glycerin, 1% Triton X-100). Extracts were cleared by centrifugation and total cellular protein was quantified using a protein determination kit (Bio-Rad). Protein solution (80 µg) was mixed with the luciferase reagent (470 µM luciferin, 530 µM ATP, 20 mM tricin, 1·07 mM (MgCO3)4 . Mg(OH)2 . 5H2O, 2·67 mM MgSO4, 0·1 mM EDTA, 33·3 mM DTT, pH 7·8). The relative light units were determined during 10 s in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Each transfection was repeated at least three times in duplicate with at least three different DNA preparations. The average values with the standard deviation were plotted.

{blacksquare} Northern blot analysis.
HepG2 cells (2·5x106) were seeded onto 10 cm tissue culture dishes 24 h prior to transfection. Forty- two hours after lipofection with 10 µg plasmid DNA, cells were washed three times with PBS, lysed and total cellular RNA was isolated with the RNeasy Total Purification kit as recommended by the supplier (Qiagen). Total RNA (20 µg) was separated in a 1% agarose/6·5% formaldehyde gel and blotted to a nylon membrane (Hybond-N+; Amersham). The membrane was prehybridized at 42 °C in 5x SSPE (50 mM NaH2PO4 . H2O, pH 7·4, 600 mM NaCl, 5 mM EDTA), 2x Denhardt's solution, 50% formamide, 1% sarcosyl, 0·1% SDS, 100 µg/ml salmon sperm DNA for 8 h. For hybridization, 0·2 vol. 50% dextran sulfate solution and 106 Cerenkov c.p.m./ml of a 32P- labelled luciferase or ß-actin-specific probe (specific radioactivity, 108–109 Cerenkov c.p.m./µg DNA) were added and the membrane was incubated at 42 °C overnight. Subsequently, the membrane was rinsed twice for 20 min in 2x SSC (300 mM NaCl, 30 mM sodium citrate, pH 7·0), 0·5% SDS and once for 15 min in 0·1x SSC, 0·1% SDS and was autoradiographed.

{blacksquare} Nuclear transcription run-on analysis.
Nuclear transcription run-on analysis was carried out by methods described previously (Farrell, 1993 ). Briefly, HepG2 cells were transfected as described in the Northern blot section. Forty-two hours later, nuclei were prepared and nascent RNA transcripts were elongated in the presence of [{alpha}-32P]UTP. Plasmid DNA fragments (2 µg) were immobilized on a nylon membrane (Hybond- N+) and hybridized with the purified, radioactively labelled RNAs. Plasmid DNA fragments containing the CMV promoter, the respective HBV sequence in the sense or antisense orientation, the internal ribosomal entry site (IRES) element and the luciferase gene were prepared by cleaving plasmids pHBV1638–1783s, pHBV1783–1638as, pHBV1374–1783s, pHBV1783–1374as, pHBV1465–1686s and pHBV1686–1465as with MluI and XhoI. Control plasmids pCMV-NCRluc and pRc/CMV were both digested with MluI and XhoI. Plasmid pAL41 (Alonso et al., 1986 ) was digested with PstI to remove the inserted ß-actin gene. The resulting plasmid fragments were separated by agarose gel electrophoresis and purified using the QiaEx II gel extraction kit (Qiagen).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
A regulatory element inhibiting gene expression is located between nt 1038 and 1783 of the HBV plus-strand
To investigate the existence of regulatory elements on the plus- strand of the hepatitis B virus, we generated two constructs containing nt 1038–1783 of the HBV sequence in either of the two orientations between the CMV early enhancer/promoter complex and an IRES with stop codons in all three reading frames followed by the luciferase reporter gene (Fig. 1 ). The plasmids were designated pHBV1038–1783s, containing the HBV sequence in minus-strand (sense) orientation and pHBV1783–1038as, containing the HBV sequence in plus-strand (antisense) orientation. Expression of these plasmids should lead to an HBV/luciferase fusion mRNA transcript but not to an HBV/luciferase fusion protein, because of the stop codons in the IRES element. Thus, synthesis of the regular luciferase protein should not be affected. The inserted HBV sequence contains, in minus- strand orientation, the X ORF from nt 1374 to 1783 encoding the first 136 amino acids (aa) of the HBx protein. Deletion analyses have already revealed that this HBx fragment is unable to act as a transcriptional activator (Kim et al., 1993 ; Murakami et al., 1994 ; Renner et al., 1995 ). Thus, a possible influence of a synthesized HBx protein on luciferase gene expression via transactivation of the CMV promoter can be excluded.



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Fig. 1. Schematic representation of the structure of the HBV deletion plasmids. Various fragments of the HBV genome were inserted in HBV minus-strand (sense) orientation or plus-strand (antisense) orientation as described in Methods. The names of the plasmids indicate the nucleotides present in the respective HBV sequence inserted in sense (s) and antisense (as) orientation. PCMV , early enhancer/promoter region of human cytomegalovirus; IRES, internal ribosomal entry site; tl stop, stop codons in all three reading frames; luciferase, luciferase gene; pA, polyadenylation signal of the bovine growth hormone gene.

 
After transient transfections with these plasmids of different liver- derived cell lines (HepG2, CCL13) and a non-hepatic cell line (HeLa), luciferase gene expression from plasmid pHBV1783–1038as was reduced in all cell lines tested compared to plasmid pHBV1038–1783s (5·3-fold in HepG2 cells, 6·7-fold in CCL13 cells and 5·9-fold in HeLa cells; Table 1 ). The inhibitory effect of the HBV element in antisense (plus-strand) orientation was verified by introducing the cloning vector pCMV-NCRluc in all three cell lines, resulting in luciferase synthesis similar to that of the sense-plasmid pHBV1038–1783s (data not shown).


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Table 1. Inhibition of luciferase gene expression by different HBV sequences inserted in the antisense orientation

 
These data indicate that the region between nt 1038 and 1783 of the HBV genome carries a regulatory element active in antisense orientation which downregulates gene expression.

Deletion analysis of the inhibitory element
To determine the minimal sequence sufficient for the inhibitory function, we generated pairs of luciferase expression plasmids, which contain different fragments out of nt 1038–1783 of the HBV sequence in minus-strand (sense) and plus-strand (antisense) orientation, resulting in a series of sense and antisense-plasmids as described in Fig. 1. Transfection of HepG2 cells, CCL13 cells and HeLa cells with these plasmids and subsequent luciferase assays revealed that 5'-truncations of the HBV sequence up to nt 1638 [plasmids pHBV1783–1374as (no. 4), pHBV1783–1402as (no. 6) and pHBV1783–1638as (no. 8)] do not affect the negative regulatory function (Table 1, columns 2, 3 and 4). In contrast, plasmids pHBV1465–1238as (no. 10) and pHBV1686–1465as (no. 12), which carry deletions at the 5'- end and at the 3'-end of the HBV sequence, exert no inhibitory effect (Table 1, columns 5 and 6, respectively). In this respect, we were able to delimit the region essential for inhibitory function between nt 1783 and 1638 (plasmid no. 8). That the inhibitory potential of the minimal specific region within plasmid pHBV1783–1638as depends on the HBV sequence was confirmed in cell culture experiments with plasmid pCMV206luc, which carries a non- coding, non-regulatory 206 bp sequence of pUC19 between the CMV promoter and the IRES-luc element. Luciferase gene expression from this plasmid was shown to be similar to the sense-plasmid pHBV1638–1783s and the cloning vector pCMV-NCRluc (data not shown).

In the plasmid context used here, the functional HBV sequence from nt 1638 to 1783 contains, in both orientations, three ATG codons which can function as initiation sites for the translation of three peptides of 79, 35 and 25 aa in length. To determine whether these peptides would be synthesized and would influence luciferase gene expression in trans, we performed in vitro transcription/translation assays with plasmids pHBV1638–1783s and pHBV1783–1638as, respectively, using 35S-labelled methionine and leucinol to avoid post-translational removal of the methionine residue. Autoradiography of the separated proteins revealed only a single band of 65 kDa corresponding to the molecular mass of the luciferase protein (data not shown). Thus, these data suggest that the observed inhibitory effect is not based on a protein-mediated mechanism in trans.

The truncated HBV enhancerII/core promoter complex located within the identified element does not affect its inhibitory function
The enhancerII/core promoter complex of HBV is present in part within the inhibitory element from nt 1638 to 1783 (Yee, 1989 ; Yuh et al., 1992 ; see also Fig. 1 ). Although the transcription initiation sites of the precore/core RNA at nt position 1785 (Zhang & McLachlan, 1994 ), 1794 (Chen et al., 1995 ; Hiraga et al., 1994 ) and 1815 (Hiraga et al., 1994 ) are missing, it remains possible that transcripts initiating at the core promoter site of plasmid pHBV1783–1638as would be able to hybridize with the complementary HBV/luc-mRNA, resulting in a reduced translation rate of these mRNA molecules. We therefore analysed whether the enhancerII/core promoter complex is active and may contribute to the observed inhibition of luciferase gene expression. For this purpose, plasmids were constructed that do not contain any promoter element upstream of the IRES-luc cassette [p({Delta}CMV)NCRluc; Fig. 2a ] or carry only the HBV DNA nt 1638–1783 spanning parts of the enhancerII/core promoter region [p({Delta}CMV)HBV1638–1783; Fig. 2a ]. In transfection experiments using HepG2 cells, CCL13 cells and HeLa cells, both plasmids revealed a similar, extremely weak expression of the luciferase gene (Table 2, columns 2 and 3, respectively) compared to plasmid pCMV-NCRluc which carries the CMV early enhancer/promoter complex 5' to the luciferase gene (Table 2 , column 1). If the enhancerII/core promoter complex had been active, a higher expression would have been expected from the plasmid p({Delta}CMV)HBV1638–1783.



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Fig. 2. Plasmid constructs. (a) Schematic representation of luciferase expression plasmids used for evaluating the HBV enhancerII/core promoter activity. HBV, HBV genome sequence from nt 1638 to 1783 containing the enhancerII/core promoter complex (core promoter) in part; {Delta}HBV, deleted HBV genome sequence; {Delta}CMV, deleted early enhancer/promoter region of human cytomegalovirus. (b) Diagram of luciferase expression plasmids under control of the SV40 promoter complex. PSV, simian virus 40 early enhancer/promoter complex; 1638HBV1783 , HBV genome sequence from nt 1638 to 1783 integrated in minus-strand direction; 1783HBV1638, HBV genome sequence from nt 1783 to 1638 integrated in plus-strand direction. (c) Schematic representation of luciferase expression plasmids containing the inhibitory HBV element in both directions, respectively, either upstream of the CMV promoter (pHBV1638–1783CMVluc, pHBV1783–1638CMVluc) or downstream of the luciferase gene (pCMVlucHBV1638–1783, pCMVlucHBV1783–1638). For explanation of additional abbreviations see legend to Fig. 1.

 

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Table 2. Luciferase gene expression by plasmids lacking a promoter element or carrying only parts of the HBV enhancerII/core promoter region

 
In addition, RNase-protection experiments were performed to analyse whether RNA was transcribed from the truncated enhancerII/core promoter complex. No HBV-specific transcripts could be detected in these experiments (data not shown). These results show that the truncated enhancerII/core promoter complex is either not active or only active to a minimal degree and therefore not able to account for the activity of the identified regulatory element.

To clarify whether the inhibitory effect is triggered by an unspecific element created by fusing the HBV sequence to the CMV enhancer/promoter complex, we examined the regulatory potential of the HBV element in a different promoter context.

In plasmids pSV(HBV1638–1783) and pSV(HBV1783–1638) the identified element is inserted in either sense or antisense orientation, downstream of the simian virus 40 (SV40) early enhancer/promoter complex, respectively (Fig. 2b ). An inhibitory effect on luciferase gene expression after transfection of liver and non-liver cells (HepG2, CCL13, HeLa) was observed with the antisense-plasmid pSV(HBV1783–1638) compared to the sense-plasmid pSV(HBV1638–1783) (Table 3 , column 1). These results demonstrate that the downregulatory effect of the HBV element is promoter- independent.


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Table 3. Inhibition of luciferase gene expression by the identified HBV element in a different promoter context

 
The inhibitory element acts in a position-dependent manner
To elucidate whether the minimal functional element from nt 1783 to 1638 acts in a position-dependent manner, we subcloned this sequence in both orientations upstream of the CMV early enhancer/promoter complex, resulting in plasmids pHBV1638–1783CMVluc and pHBV1783–1638CMVluc (Fig. 2c ). After transcription of these plasmids, the HBV sequence would not be a component of the luciferase mRNA molecules. To elucidate the inhibitory potential of the HBV element in this position, we performed transfection experiments with HepG2 cells, CCL13 cells and HeLa cells. In subsequent luciferase assays, no inhibition of gene expression with the antisense-plasmid pHBV1783–1638CMVluc compared to the sense-plasmid pHBV1638–1783CMVluc (Table 4 , column 2) and the control vector p206CMVluc containing a non-functional 206 bp fragment of plasmid pUC19 upstream of the CMV promoter (data not shown) was detectable.


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Table 4. Position dependence of the inhibitory effect of HBV fragment 1783–1638

 
In addition, we generated plasmids pCMVlucHBV1638–1783 and pCMVlucHBV1783–1638, which carry the HBV sequence in either direction downstream of the luciferase gene (Fig. 2c ). In these cases, the HBV element is located on the luciferase mRNA. Luciferase assays demonstrated that compared to the sense-plasmid pCMVlucHBV1638–1783, plasmid pCMVlucHBV1783–1638, which contains the functional element in the proper antisense orientation, exerts no downregulation of luciferase gene expression in the cell lines tested (Table 4, column 3). Using the control plasmid pCMVluc206, which carries the 206 bp fragment of plasmid pUC19 downstream of the luciferase gene, similar results were obtained (data not shown).

From these data, we conclude that the inhibitory element not only acts in an orientation-dependent manner, but also in a position- dependent manner.

The inhibitory element impairs gene expression via a reduced mRNA steady-state level
To investigate the mechanism by which the HBV element mediates its inhibitory function, we determined the level of luciferase mRNA present in HepG2 cells transfected with different sense and antisense-plasmids (pHBV1638–1783s, pHBV1783–1638as, pHBV1374–1783s, pHBV1783–1374as, pHBV1238–1465s and pHBV1465–1238as) by Northern blotting experiments. The blots were hybridized with a luciferase sequence-specific DNA probe and, to provide an internal standard for mRNA quantification, with a ß-actin DNA probe.

As shown in Fig. 3, a reduced luciferase mRNA steady- state level was obtained with plasmids containing the inhibitory element in antisense orientation compared to the respective sense- plasmids (lanes 2 and 4 compared to lanes 1 and 3, respectively). Expression of plasmid pHBV1465–1238as, that lacks the regulating sequence, revealed no decrease of the luciferase mRNA level (Fig. 3 , lane 6 compared to lane 5). The observed differences in mRNA amounts suggest that the inhibitory element acts either as an atypical DNA silencer or via destabilization of mRNA, rather than via a post-transcriptional mechanism affecting translational initiation or elongation.



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Fig. 3. Effects of the inhibitory HBV element on luciferase mRNA steady-state level. Forty-two hours after transfection of HepG2 cells with the indicated plasmids, RNAs were extracted and analysed by Northern blot experiments. After hybridization with a 32P- labelled luciferase probe, autoradiography with Amersham Hyperfilm-MP was carried out at -75 °C for 24 h, using an intensifying screen (upper panel). The blot was stripped and rehybridized with a ß-actin-specific probe to control for RNA loading (lower panel).

 
The inhibitory element reduces the mRNA steady-state levels without modulating transcriptional initiation rates
The Northern blot results prompted us to elucidate whether the reduced luciferase mRNA steady-state level is due to a reduced transcription initiation rate or to a lower stability of the respective transcripts. For this purpose, nuclear run-on analyses were performed after transfection of HepG2 cells with different sense- and antisense- plasmids. The labelled RNA was hybridized to an excess of immobilized plasmid fragments containing only the CMV promoter, the HBV sequences in sense and antisense orientation, respectively, the IRES element and the luciferase gene (except fragments of pRc/CMV and pAL41) (for details see legend to Fig. 4 and Methods).



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Fig. 4. Nuclear run-on assays. HepG2 cells (5x106 ) were transfected with 10 µg plasmid (A, pHBV1638–1783s; B, pHBV1783–1638as; C, pHBV1374–1783s; D, pHBV1783–1374as; E, pHBV1465–1686s; F, pHBV1686–1465as). At 42 h post- transfection, nuclei were isolated and newly initiated mRNA was labelled to a high specific activity with [{alpha}-32P]UTP. The radiolabelled RNA was hybridized to different DNA fragments immobilized on a nylon membrane. The immobilized fragments 1+2, 6+7 and 8+9 contained the CMV promoter, the HBV fragments nt 1638–1783 (1), nt 1783–1638 (2), nt 1374–1783 (6), nt 1783–1374 (7), nt 1465–1686 (8) and nt 1686–1465 (9), the IRES element and the luciferase gene. DNA fragment 3 contains the ß- actin gene of plasmid pAL41. Fragment 4 is derived from plasmid pCMV- NCRluc and spanned the CMV promoter, the IRES element and the luciferase gene, whereas fragment 5, as a negative control, contained only the CMV promoter of plasmid pRc/CMV.

 
As shown in Fig. 4, no significant difference in the amount of hybridization to the respective DNA fragments between the transcripts synthesized from the sense- and antisense-plasmids was detectable (compare bands A1 and B2, C6 and D7, and E8 and F9, respectively). Surprisingly, only weak bands were detected with a ß-actin-specific DNA fragment to determine the internal ß- actin transcription initiation rate (Fig. 4, A3–F3). Nevertheless, the hybridization signals are specific, since only a slight signal was observed when labelled RNA was used to hybridize to the pRc/CMV vector DNA fragment (Fig. 4, A5–F5). These results demonstrate that the reduced luciferase mRNA levels obtained in Northern analyses of cells transfected with plasmids containing the HBV element in antisense orientation are based on a lower stability of these transcripts rather than on reduced transcription initiation rates. Therefore, we conclude that the identified HBV element within nt 1783–1638 acts as an RNA- destabilizing factor.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The minus-strand of HBV encodes all HBV gene products known so far. Transcription from this DNA strand has been analysed extensively. Limited information is available about transcription from the complementary plus-strand (Standring et al., 1983 ; Zelent et al., 1987 ) and about regulatory elements on this strand (Velhagen et al., 1995 ; Shimoda et al. , 1998 ). In this study, we have presented data demonstrating the existence of a novel cis-regulatory element on the plus-strand of HBV. This element reduces gene expression of a subsequent luciferase reporter gene in a plus-strand orientation- dependent manner. The observed effect is not restricted to liver cells, since inhibition of luciferase gene expression was detectable in liver- derived cell lines (HepG2 cells, CCL13 cells) as well as in a cervix carcinoma cell line (HeLa cells). Deletion analyses revealed that the sequence essential for the inhibitory function of the element is located between nt 1783 and 1638 of the HBV genome. This region contains most of the HBV enhancerII/core promoter complex that regulates transcription of the core and polymerase ORF located on the minus-strand (Yee, 1989 ). Thus, in our experimental system, one can speculate that a truncated, but still functional core promoter would lead to RNA molecules complementary to the luciferase mRNA. This would result in hybridization of these RNAs and, from that, possibly in a reduction in luciferase protein synthesis. However, there are several points arguing against the enhancerII/core promoter being active in our system. Firstly, the transcription initiation sites of the core promoter were shown to be located at nt 1785 (Zhang & McLachlan, 1994 ), 1794 (Chen et al., 1995 ) and 1815 (Hiraga et al., 1994 ). Since the investigated HBV element does not extend beyond nt 1783, synthesis of a core promoter-specific RNA should not take place. Secondly, the core promoter is highly liver cell-specific (Honigwachs et al., 1989 ; Yuh et al., 1992 ; Zhang & McLachlan, 1994 ), yet the inhibitory effect was observed both in liver and non-liver cells to the same extent, suggesting that the core promoter is not involved. Thirdly, in transfection experiments, we were able to demonstrate that the truncated enhancerII/core promoter complex within the investigated HBV sequence revealed a very low activity compared to the activity of the CMV promoter (Fig. 2a; Table 2, compare columns 3 and 1). As well as this, Lser et al. (1996 ) described only a weak core promoter activity in HepG2 cells compared to the activity of the CMV promoter. In non-liver HeLa cells and CV1 cells, core promoter activity was diminished to a higher degree (Löser et al., 1996 ). The hybridization of complementary mRNA molecules occurs in a one-to-one ratio (Nellen & Lichtenstein, 1993 ), i.e. one HBV core promoter transcript can inhibit only one CMV promoter transcript at any one time. Thus, it is unlikely that the low numbers of HBV core promoter transcripts could be responsible for the observed inhibitory effect by complementing most of the CMV promoter transcripts, which are present at a much higher level. Indeed, in RNase protection experiments (data not shown), RNA transcribed from the truncated HBV enhancerII/core promoter was below the threshold of detectability, indicating no or a very weak core promoter activity.

Nuclear run-on analyses revealed that the transcription initiation rate of RNA molecules carrying the regulatory element is not affected (Fig. 4). However, as shown in Northern experiments, the overall level of cellular RNA containing the element in the proper orientation is significantly reduced (Fig. 3). This suggests that the negative regulatory element acts at a post- transcriptional level, for example by reducing mRNA stability. This hypothesis is supported by the observed position-dependency of the negative regulatory element. To mediate its function, the active sequence must be included in the transcript since no effect was observed when it was placed upstream of the CMV promoter (Fig. 2c ; Table 4, column 2). This would be in agreement with the usual definition of a silencer element (Brand et al., 1985 ).

Post-transcriptional gene regulation is predominantly facilitated by regulation of RNA stability (for review, see Bellman & Parker, 1995 ; Ross, 1996 ; Sachs, 1993 ). Common RNA-destabilizing elements are represented by so-called ARE elements which contain one to multiple adenylate/uridylate-rich motifs or in general a high degree of adenines and uracils. ARE elements are exclusively located in the 3'-noncoding regions of mRNAs (for review, see Chen & Shyu, 1995 ). The amount of adenines and uracils of the RNA element described here is less than 55% and there are no AU-rich clusters. In addition, the inhibitory element is not functional when located in the 3'-noncoding region of the transcript (Fig. 2c; Table 4, column 3). Thus, it is unlikely that this element acts via an `AU element-like' mechanism. Recently, various cellular and viral RNA elements modulating mRNA stability have been identified within the protein coding region. RNAs of the c-myc (Wisdom & Lee, 1991 ), the c-fos (Wellington et al., 1993 ) and the ß-tubulin (Yen et al., 1988) ORF carry destabilizing sequences just as the pol -env region of human T-lymphotropic virus 1 (Saiga et al. , 1997) , the L2 coding region of the human papillomavirus type 16 (Sokolowski et al., 1998) and the MAT{alpha}1-mRNA (Parker & Jacobson, 1990) do. These elements mediate their function via different mechanisms. For example, degradation of the Mat{alpha}1-mRNA is based on the presence of rare codons in the coding region, whereas degradation of c-myc mRNA, c-fos mRNA and ß-tubulin mRNA is due to the start of translation (Hennigan & Jacobson, 1996 ; Herrick & Ross, 1994 ; Parker & Jacobson, 1990 ; Schiavi et al., 1994 ). However, it remains to be investigated whether one of these mechanisms or a completely different one is responsible for the inhibitory function of the plus-strand element. Beside this, it would also be possible that the RNA molecules containing the inhibitory element will be prematurely destroyed in the nucleus.

Interestingly, the identified regulatory element acts not only in a position-dependent manner but is also orientation-dependent. This is due to the occurrence of an inhibitory effect of this element only if it is inserted in plus-strand orientation. To date, transcriptional regulatory RNA elements, acting both in an orientation- and position- dependent manner, have been rarely detected. Recently, Young & Korsmeyer (1993) identified a position-dependent negative- regulatory element in the bcl-2 5'-untranslated region with distinct orientation-dependent subfragments. However, although it seems to be an RNA element, they do not commit to whether this NRE is functional at an RNA or a DNA level.

We have clearly shown the existence of a negative regulatory RNA located on the plus-strand of HBV acting both in an orientation- dependent and a position-dependent manner. However, the relevance of this element for the life-cycle of HBV or its host cell is currently not known. One can speculate that the regulatory function of this element minimizes the presence of stable ORF6-derived mRNA molecules, whereas the presence of these mRNAs would lead to a possibly toxic ORF6 protein. This might subsequently result in death for the host cell. Another possibility might be that stable plus-strand-derived mRNA molecules would interact with HBV minus-strand mRNAs, which all overlap the identified regulatory region, resulting in a decreased level of HBV replication or protein expression. If so, the virus may have developed strategies during evolution to reduce the amount of plus-strand mRNA molecules, for example by evolving a plus-strand-specific RNA- destabilizing element, like the one that is described here.

However, the importance of this inhibitory element can only be evaluated by investigating its function in the natural context of the HBV genome. To this end, the behaviour of the virus and its host cells must be investigated in the presence and absence of the inhibitory element. For this approach, the regulatory function can be inactivated by mutation of relevant nucleotides. However, the complexity of this region (i.e. the presence of the X and polymerase ORF, the enhancerII region and other minus-strand-specific cis-regulatory elements) will complicate such an analysis.


   Acknowledgments
 
We thank S. Alonso for providing plasmid pAL41. We would also like to thank Silva Andric for her technical assistance and Christine Schommer for sequencing all cloning products. We are grateful to Robin Steigerwald and Brian Salmons for critical reading of the manuscript. This work was supported by grants from the Deutsche Stiftung f ür Krebsforschung to P.H.H.


   Footnotes
 
b Present address: Max von Pettenkofer-Institut f ür Hygiene und Mikrobiologie, Ludwig Maximilians-Universität München, Feodor-Lynen-Straße 25, D-81377 M ünchen, Germany.

c Present address: Bavarian Nordic Research Institute, Institut für Virologie, Veterin ärmedizinische Universität Wien, Am Veterinä rplatz 1, A-1210 Wien, Austria.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 8 June 1999; accepted 15 June 1999.