Regulation of hepatitis B virus S gene promoter in transfected cell lines

162, 362-368

VIROLOGY

(1988)

Regulation

of Hepatitis

B Virus S Gene Promoter

OURIEL FAKTOR, TALI DE-MEDINA, Department

of Virology, Received

The Weizmann June

18, 1987;

Institute accepted

AND of Science, September

in Transfected

Cell Lines

YOSEF SHAUL’ Rehovot

76 100, Israel

24, 1987

Hepatitis B virus (HBV) contains an enhancer element that activates the viral core and X gene promoters. To investigate the transcriptional regulation of the viral S gene promoter, we transfected SK-Hepl cells with circularized forms of HBV DNAs and their enhancerless mutants. We have found that expression of the S gene, determined by measurement of the appearance of HBsAg in the media and by RNA analysis, is to a large extent enhancer-dependent. This observation was further confirmed by analysis of a series of plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of the S gene promoter and the HBV enhancer element. Interestingly, in contrast to its behavior in SK-Hepl cells, the S gene promoter is highly active in Alexander cells, in the absence of the enhancer element. This implies that activity of the S gene promoter is cell-type specific. o t989 Academic PWW, IIIC.

INTRODUCTION

study of the S gene (Gough and Murray, 1982; Pourcel et al., 1982; Malpiece et al., 1983; Standring et a/., 1984). However, we believe that this experimental approach is not suitable for the study of the viral transcriptional regulatory elements for the following reasons: (I) The strong promoter and enhancer elements often used to activate the desired selective marker gene may also activate in cis the cotransfected S gene. (II) This method allows insertion of only a relatively low copy number of viral DNA into the host chromosome, as opposed to a higher copy number of extrachromosomal viral DNA expected to be found in a replicative system. Since the anticipated cellular factors regulating the transcription of the S gene are believed to be limited in amount, differences in copy number of viral DNA may be of great significance. To overcome these limitations, and to mimic as closely as possible the scenario of natural infection we transfected cells with circularized HBV DNA in unselected cell culture. Under these conditions, high and efficient expression of HBsAg under the control of the genuine S gene promoter was achieved. We report here that the deletion of the HBV enhancer element resulted in complete abolishment of S gene mRNA and of HBsAg production. This enhancerdependent activity of the S promoter was further demonstrated in a heterologous system using the chloramphenicol acetyltransferase (CAT) gene as a test gene. Interestingly, in Alexander cells, known to express HBsAg constitutively, high activity of the S gene promoter was found in the absence of the enhancer element. We propose that the S promoter is regulated by the viral enhancer element and by an additional element positioned at or adjacent to it.

Hepatitis B virus (HBV) is a small DNA virus of humans and is considered to be the etiological agent of a major form of hepatitis. Considerable progress has been made recently in elucidating the structure of the HBV virion and its genome (for a review, see Tiollais et al., 1985). The virion is a 42-nm particle whose outer coat is composed mainly of a single protein, the hepatitis B surface antigen (HBsAg), in both glycosylated and nonglycosylated forms (~24 and gp27). Detailed examination of the surface antigen has indicated that it is composed of two additional pairs of proteins: p31 and gp37, p39 and gp42. These proteins are derived from a large open reading frame (ORF) which consists of the S region, which is preceded by the inphase pre-S-l and pre-S-2 reading frames. Expression of the S ORF (the S gene) has been achieved so far in a variety of human and non-human cell lines derived from a number of different tissues. Detailed characterization of the S gene mRNA (the 2.1 -kb message) shows that transcription initiates within the pre-S region with three major start sites spanning some 30 bp around the EcoRl site (Standring et a/., 1984). Despite the extensive mapping that has been done of S gene mRNA initiation sites, nothing is known about the regulatory elements which modulate the S gene promoter. Stable insertion of HBV sequences into tissue culture cells frequently leads to production and secretion of HBsAg and has served as a convenient tool for the ’ To whom 0042.6822188 Copyright All rights

requests

for reprints

$3.00

0 1988 by Academic Press, Inc.. of reproduction I” any form reserved.

should

be addressed. 362

HBV

MATERIALS

S GENE

AND METHODS

Cell growth HeLa, PLC/PRF/5 (Alexander) and SK-Hepl cell lines were cultured in Dulbecco’s modified Eagle’s minimum essential medium (GIBCO) containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 Kg/ml streptomycin.

Construction

of plasmids

The cloned HBV genome used in our study is the 3.2-kb HBV of Valenzuela eta/. (1980). Integrated HBV DNA was isolated previously from the Alexander cell genome and was designated clone 26 (Shaul et al., 1984). All DNA constructs were prepared by standard recombinant DNA techniques (Maniatis et al., 1982). The circularized HBV DNAs were prepared by digestion of the plasmids by enzymes that remove the vector DNA and subsequently self-ligate in a reaction mixture containing l-2 pg DNA/ml. The efficiency of the circularization was tested using agarose gel electrophoresis by detection of the appearance of a new DNA fragment after cleavage with the Xbal restriction enzyme. The circularized DNA was passed through an RPC-5 column before being used for transfection of cell cultures.

Cell transfection

and CAT and HBsAg assays

Cells were transfected and CAT assays were performed following the procedure described previously (Gorman et al., 1982). HBsAg was determined using a commercial radioimmunoassay kit (Abbott AUSRIA II). DNA uptake by the two most common cell lines, Alexander and SK-Hepl cells, was measured by comparing the amounts of extrachromosomal DNA present 48 hr after transfection. The uptake of DNA by Alexander cells was about 50% that of SK-Hepl cells.

Preparation

and analysis of RNA

For each DNA construct, RNA was prepared from five lo-cm tissue culture dishes, about 40-48 hr (using the CAT gene constructs) and 50-60 hr (using the circularized S gene constructs) post-transfection, by the guanidium thiocyanate method (Chirgwin et a/,, 1979). The RNase mapping was carried out using a method previously described (Zinn eta/., 1983; Melton et al., 1984). The primer extension analysis procedure, using a single-stranded oligonucleotide of CAT gene, was as described previously (Walker et a/., 1983). For slot blot analysis, about 10 rg of each RNA sample was denatured by formaldehyde before being applied onto a nitrocellulose filter. Riboprobes were used for

363

PROMOTER

hybridization. Filters were subjected ment before exposure to X-ray film.

to RNase

treat-

RESULTS The HBV enhancer element expression of the S gene

is required

for

We first studied the regulation of the promoter of the S gene by transfection of tissue culture cells with circularized HBV DNA prepared from the pH3HBV plasmid, which contains an intact HBV DNA of the adw subtype (Shaul et a/., 1984). In this plasmid, the HBV DNA was cloned into the HindIll site of pBR322 at its unique Haell (1440) site, using HindIll linkers (Fig. 1). pH3HBV was digested by HindIll enzyme and ligated under conditions favoring self-ligation. SK-Hepl , a human hepatoma cell line devoid of HBV DNA (Chang et a/., 1983), was transfected with the circularized HBV DNA, and the media was analyzed every second day for the presence of HBsAg, using an RIA commerical kit (Fig. 2A). High levels of HBsAg synthesis were obtained, with maximal production at the sixth to seventh day after transfection. To test whether this HBsAg production is regulated by the previously defined HBV enhancer element, we constructed the pH3HBVAE plasmid in which the EcoRV (1043)-Sphl (1235) viral fragment, known to contain the HBV enhancer sequences (Shaul et al., 1985) was removed (Fig. 1). This construct gave about 50-fold less production of HBsAg than the parental plasmid, when transfected into SK-Hepl cells (Fig. 2A). This observation strongly suggests that the enhancer element is crucial for HBsAg synthesis. However, since the enhancer element is a portion of the coding region of the viral polymerase gene (Fig. 1A), the enhancerless mutant lacks also an intact polymerase gene, a situation in which a potentially infectious DNA will be transformed into a noninfectious one. To confirm that the synthesis of HBsAg is regulated by the enhancer element and not by the infectivity of the DNA, we used an incomplete HBV DNA, clone 26, which has been previously isolated from the genome of the Alexander cells @haul et a/., 1984). This DNA contains about 80% of the viral genome, with the intact S gene and its promoter and the viral enhancer element, but does not contain the core, the X, and the intact polymerase genes @haul et al., 1984; Ziemer et al., 1985). A 3.8-kb Pstl fragment which contains 2.4 kb of integrated HBV sequence and 0.7 kb of host cellular sequences at both ends of the viral-host junctions was subcloned to generate the p3.8AL (Fig. 1). As observed with prototype HBV DNA, the circularized 3.8-kb Pstl fragment directed the synthesis of high

364

FAKTOR,

DE-MEDINA,

AND

SHAUL

B

TATA NFI

IP

pH,HBVAE

RPPS

P

s

T

,

?SPR

R

AS

TATA NFI IP

wv2) p2.4AI

sv40

(1467)

SVT TATA NFt IP

FIG. 1. Structures of HBV genome and DNA fragments used to transfect SK-Hep cells for HBsAg production. (A) The HBV genome and its transcripts with the four major open frames (ORF) and their map position are shown. Gene S encodes for the viral surface antigen, gene C encodes for the core antigene, P and X designate the ORFs that encode for the polymerase and X proteins, respectively. Also shown is the position of the enhancer element and of the S gene promoters TATA and internal promoter (IP), and the C gene promoter (CP), and the X promoter (XP). (B) The EcoRl 3.2-kb HBV DNA (Valenzuela et al., 1980) was circularized and relinearized by Haell enzyme (H,-1440) and recloned in the HindIll (H3) site of pBR322, using the HindIll linker to construct the pH3HBV. The EcoRV (EB-1 043) and Sphl (Sp 1235) fragment containing the HBV enhancer element (E) was deleted for construction of pH,HBVAE. (C) Clone AL26, derived from integrated HBV DNA in Alexander cells as previously described (Shaul et a/., 1984; Ziemer et a/., 1985) was digested by Pstl (P) enzyme and the 3.8 Pstl fragment containing the integrated HEW sequence was recloned in SP64 plasmid for construction of the p3.8AL (Berger and Shaul, 1987). In p3.8ALAE the enhancer element (E) of p3.8AL was removed by the restriction enzymes Sful (St) and Sphl (Sp). For construction of p2.4ALSVT the 2.4-kb Smal (S) fragment containing the entire integrated HBV sequence isolated from clone AL26 was recloned in the Small site of the SP64 plasmid in which the SV40 Rsal (Rs) fragment (1467 to 3072) was inserted at the /WI1 (Pv) site. In this clone, the polyadenylation site P(A) of p3.8AL plasmid was replaced by that of SV40. The thin lines represent the host sequences of the integrated HBV DNA, the thick lines the HBV sequences, and the S gene is shown by a black arrow. The dashed region represents the preS1 and preS2 positions of the gene; the open box, the SV40 sequences; and the dotted line, the SP64 DNA fragment. The S gene internal promoter (IP) next to the EcoRl (R0/3221) site is shown by a small arrow. The positions of the TATA box, the nuclear factor I binding site (NF-I) which we have recently defined (Shaul et a/., 1986), the polyadenylation site P(A) and the enhancer element (E and black box) are shown. The Xbal (Xb) sites used to confirm the circularized forms of HBV DNA are also shown. Xs and XI are the short and the long arms of the lambda vector DNA, respectively.

levels of HBsAg in SK-Hepl cells (Fig. 28). Therefore, intact and hence infectious HBV DNA is not required for HBsAg synthesis. We next removed the viral enhancer element by deletion of the Stul (1 1 15)-Sphl (1235) fragment, (p3.8ALAE, Fig. 1). In agreement with results obtained with the prototype viral DNA, this deletion resulted in almost total abolishment of HBsAg production by transfected SK-Hepl cells (Fig. 2B). It is shown, recently, that the core and the X gene promoters are activated by the viral enhancer element (Shaul et a/., 1985; Treinin and Laub, 1987). Assuming that the HBV termination and polyadenylation signals

are “leaky,” it is possible although very unlikely that the synthesis of the HBsAg is programmed by readthrough mRNAs derived from the above promoters rather than from the S promoter. To rule out this possibility, we replaced the polyadenylation signal by that of the SV40 (construct p2.4AISVT, Fig. 1). Figure 2B clearly shows that SK-Hepl cells transfected with circularized 2.4ALSVT DNA produce HBsAg in levels comparable to those obtained with 3.8AL DNA. These data suggest that the synthesis of the HBsAg is not due to a read-through transcript, an observation that will be also confirmed by analysis of the mRNA.

HBV

S GENE

A 45 7 a30Q I

15-

0

E 0

., ;.:.IJ

,

HpHEVAE

2

4

6

SO Time

2

4

3.8AIAE

-0 6

8

(days)

FIG. 2. Time kinetic of HBsAg production and secretion by SKHepl cells transfected with HBV DNAand its mutants. The plasmids described in Fig. 1 were digested by Pstl enzyme, circularized under conditions favoring self-legation of the molecules, and passed through a RPC-5 column, prior to transfection of the SK-Hepl cells. Circularized DNA (10 rg) was used for transfection by the calcium phosphate method (Graham and Van der Eb, 1973) the medium was changed daily, and the amounts of secreted HBsAg were determined every second day by radioimmunoassay. P/N 3 2.0 is considered to be positive (P, cpm in the sample; N, cpm in negative control; the negative controls ranged between 100 and 200 cpm). (A) DNA constructs based on wild-type HBV DNA. (B) DNA constructs based on the integrated form of the S gene isolated from Alexander cells.

HBV enhancer

element

activates

of the S gene promoter

365

structed a series of CAT gene-based plasmids (Fig. 4). The 820-bp Bglll (2432)~BarnHI (31) fragment of the HBV genome spanning the TATA box and the S gene internal promoter (sometime called the SV40-like promoter) region was inserted at the 5’ end of the CAT gene in the pSVOCAT vector to generate the pSPCAT plasmid. In addition, the viral Hincll (963)-BamHI (1402) fragment, known to contain the HBV enhancer element was inserted at the 3’ end of the CAT gene in plasmid pSPCAT to construct the pSPCATE (Fig. 4). In pSPCATE, the S gene promoter and the HBV enhancer element are located in positions corresponding to those of the native viral genome. After transfection of SK-Hepl cells, significant CAT activity was observed only in cells transfected with enhancer-containing pSPCATE DNA (Fig. 4) in full agreement with results obtained with the intact S gene (Fig. 2A) with SK-Hepl cells. We next removed the DNA region containing the TATA box-like promoter by deletion of the Bglll-BstXI fragment (constructs pIPCAT and plPCATE). No substantial reduction in CAT activity was obtained after transfection of cells (Fig. 4), suggesting that this ele-

the S promoter

Three major transcriptional initiation sites are known to direct the synthesis of S gene mRNA (Cattaneo et a/., 1983; Standring et a/., 1984) all positioned next to the viral unique EcoRI site. To test whether similar initiation sites are active in circularized HBV DNA, under regulation of the enhancer element, and to analyze quantitatively the S RNA, we performed RNase protection experiments (Zinn et a/., 1983; Melton eta/., 1984). SK-Hepl cells were transfected with p3.8AI, p3.8AIAE, and p2.4AISVT and RNAs were prepared after 54 hr. As a control we used RNA prepared from Alexander cells which express the S gene constitutively (Macnab et al., 1976). Figure 3 shows that cells transfected with enhancer-containing constructs express high levels of S gene mRNA which initiates from three major sites, designated a, b, and c, similar to those found in RNA from Alexander cells (lanes 2 and 6) and to that observed previously (Cattaneo et a/., 1983; Standring et al., 1984). In contrast, almost no S mRNA was found in cells that were transfected with enhancerless plasmid (lane 4). These data strongly suggest that the HBV enhancer activates the genuine S promoter in SK-Hepl cells. The activity specific

PROMOTER

is cell-type

To verify the observation that the S gene promoter is under control of the enhancer element, we con-

Xhol 129 FIG. 3. Mapping of S gene transcriptional initiation sites, To map the S gene transcription initiation sites we used the RNase mappig technique (Zinn et a/., 1983; Melton et a/., 1984). The Xhol (2883129) restriction fragment containing the S gene promoter region was isolated from the integrated form of HBV sequences p3.8AL and subcloned in pSP6 for preparation of the riboprobe. RNA samples were prepared from Alexander cells (lanes 2 and 6) from SK-Hepl cells (lanes 1 and 7) and from SK-Hepl cells transfected with circularized HBV DNA of the following plasmids: p3.8AL (lane 3), p3.8ALAE (lane 4) and p2.4ALSVT (lane 5). RNA (5 pg) from Alexander cells or from transfected SK-Hepl cells (20 pg) was mixed with lo6 cpm of the probe and subjected to RNase treatment and analysis on a sequencing gel. End-labeled Mspl-digested pBR322 DNA was used as a size marker(M). The three major initiation sites are designated a, b, and c and their corresponding positions in HBV DNA are shown.

366

FAKTOR,

DE-MEDINA,

AND

SHAUL SK

AL

H

35

1.2

25

41

1.3

30

35

0.3

@CAT

pIpcAT plPCATE

Inc----I

k----y>

_._-_ j\ -.-.___ I

1 E

4

and activity of CAT-based expression vector constructs. The S gene promoter complex from Bglll (89-2433) to BamHl FIG. 4. The structure (E-30) was inserted next to the CAT gene (open arrow) in the pSVOCAT (Gorman eta/., 1982). In pIPCAT, only the &Xl (Bx-2912)~BarnHI (B-30) fragment known to contain the S gene internal promoter (Cattaneo at al., 1983; Standring eta/., 1984) was inserted. The relative positions of the TATA box sequences, the S gene promoter NF-I binding site (Shaul et a/., 1986) and the internal promoter (IP) are shown. The HBV enhancer element(E) was inserted by ligation of the HBV Hincll (H,-963)-BamHI (B-1403) fragment to the BarnHI site of the pSVOCAT at the 3’end of the SV40 transcription termination signal (broken line) to construct the pSPCATE and the plPCATE plasmids. HeLa (H) cells and the hepatoma cell lines SK-Hepl (SK) and Alexander (AL) were transfected with 5 pg of the indicated plasmids and with pSV&AT and pSVOCAT (Gorman et a/. 1982). The CAT activity was determined after 48 hr. The values of CAT activity reported are relative to that for pSV,CAT taken to be 100%. The numbers are the average value of five experiments.

ment is not required for S gene promoter activity and that the enhancer element activated the S gene promoter. Alexander cells cnstitutively express high levels of HBsAg (Macnab eta/., 1976). It was, therefore, of obvious interest to study the regulation of the S gene promoter in this cell line. The intact S gene cannot be used for this study, since the transiently synthesized HBsAg will be masked by the massive production of this protein by the resident integrated HBV DNA. Therefore, fused S promoter-CAT gene constructs were used (Fig. 4). Unexpectedly, plasmids which do not contain the enhancer element gave rise to high levels of CAT activities in Alexander cells. Analysis of RNA prepared from the transfected cell lines, by the slot blot technique, clearly demonstrated that a high level of CAT RNA was produced in Alexander cells transfected with enhancerless plasmid (Fig. 5). Furthermore, to rule out the possibility that a cryptic nonrelevant promoter is responsible for the production of CAT-specific RNA in Alexander cells, we mapped the 5’ end of the CAT RNA by primer extension technique. The predicted three major initiation sites were found (compare Figs. 5A and 3). These results strongly suggest that, in contrast to SK-Hepl cells, S gene promoter is highly active in Alexander cells in the absence of the enhancer element, although this element confers some additive effect. To further establish the cell-type specific activity of the S gene promoter we transfected HeLa cells with plasmids containing the CAT gene under the control of the promoter. We found that this promoter is poorly active in HeLa cells, even in the presence of the enhancer element (Fig. 4). The lack of enhancer activity in HeLa cells is not surprising, since we and others have previously reported that

A

B CAT

PROBE CELL

AL

ACTIN SK

AL

SK

FIG. 5. RNA analysis of hepatoma cells transfected with CAT gene under HBV transcriptional regulatory elements. (A) An end-labeled 20-mer CAT primer (Walker et a/., 1983) was annealed with 20 pg total RNA from Alexander cells transfected with pIPCAT DNA, and subjected to primer extension analysis. The autoradiogram shows the extended products analyzed on a 6% sequencing gel. Alongside, end-labeled fragments of pBR322 DNA digested by lMspl were run, to serve as a size marker (M). The transcriptional initiation sites were designated as those in Fig. 3 and their corresponding positions on the HBV genome are shown. (B) RNA samples prepared from Alexander (AL) and SK-Hepl (SK) cells transfected with the indicated plasmids were analyzed by the slot blot method. Two riboprobes were used, the CAT gene-specific probe and the actin probe, the latter to estimate the amount of RNA bound to nitrocellulose filter.

HBV

S GENE

HBV enhancer displays tissue-specific activity and is mostly active in liver cells (Shaul et al., 1985). DISCUSSION We report here that, in the SK-Hepl cell line, the HBV enhancer element is absolutely required for efficient production of HBsAg, for the first time demonstrating the role of the HBV enhancer in the context of native HBV DNA. HBV enhancer has some unique features. This element is cotranscribed with all the known viral transcripts, and also its sequence overlaps with the coding frame of the viral polymerase-reverse transcriptase. These features should be taken into consideration in elucidating its role. The HBV genome with a deletion of the enhancer element generates transcripts with the corresponding deletion. These transcripts may be either less stable or inefficiently translated, which may explain the sharp reduction in HBsAg synthesis. To confirm that this reduction in synthesis is at least in part also a result of a reduction in the activity of the S promoter, we used the fused CAT gene in the presence or absence of the viral enhancer element. In this heterologous system, the enhancer was positioned so that its sequence was not presented in the mature mRNA and obviously did not play a role in its stability. Under these conditions, the S promoter showed a strict enhancer-dependent activity in SK-Hepl cells. It was previously reported that the core and the X gene promoters are under the regulation of the enhancer element (Shaul et al., 1985; Treinin and Laub, 1987). Therefore, HBV enhancer tightly regulates all the known viral promoters. The implication of this behavior is that some additional cis or Vans regulatory elements should be involved in the modulation of the activity of the HBV promoters in order to allow their differential expression. The observation that the S promoter is highly active in the absence of the enhancer in Alexander cells further supports this assumption. The ability of a viral promoter to function in certain cell lines as an enhancer-independent promoter is reminiscent of the action of the SV40 early promoter in undifferentiated embryonal carcinoma (EC) cells (Gorman et a/., 1985). In that case it was speculated that a trans-acting factor present in undifferentiated EC cells could act so as to render the SV40 early promoter enhancer-independent. A similar mechanism explains the behavior of 293 cells which express the trans-acting Ela factor of adenovirus, and in which a number of promoters were shown to be active in the absence of enhancer elements, although they exhibit a strict requirement for such elements in other cell types (Treisman et al., 1983). Similarly, it is conceivable that the S

PROMOTER

367

promoter is activated in Alexander cells by a trans-activator of either cellular or viral origin. We have recently found that nuclear factor I (NF-I), binds next to the S gene promoter at positions 3005-3025. Although deletion of this region resulted in reduced activity of the S promoter in transfected HeLa cells (which poorly express this promoter) @haul et a/., 1986) it has only a minor effect in transfected Alexander cells (data not shown). Thus, the efficient activity of S promoter in Alexander cells is not mediated by NF-I. An additional conclusion drawn from this experiment is that the anticipated frans-activator must interact with the HBV sequence downstream from the NF-I binding site, at the region 3025 to 30 (226 bp). To test the possibility that the HBsAg, which is produced constitutively by Alexander cells but not by SKHepl cells, trans-activates the S gene promoter, we performed a cotransfection experiment in which the pSPCAT and the circularized form of 3.8AL DNA (shown to be active in HBsAg synthesis, see Fig. 2) were introduced into SK-Hepl cells. No induction of CAT activity was observed (data not shown), suggesting that HbsAg is not the trans-activator of the S promoter. However, it is also possible that this promoter is under repression and its efficient activity in Alexander cells is due to titration out of the repressor by the multiple integrated forms of the HBV DNA. Alexander cells contain at least seven independent copies of HBV DNA fragments, three of which (clones 7, 15, and 23) contain the S gene promoter region twice (Shaul et al., 1984). There are therefore at least 10 copies of the S promoter region in this cell line which can serve as a binding site for the putative repressor. To test this possibility, we performed in viva competition assays in SK-Hepl cells. Increasing amounts of a competitor DNA (a plasmid with the S gene promoter fragment) were cotransfected with the test plasmid (pIPCAT) and values of CAT activity were determined. Addition of an up to 30-fold molar excess of the competitor DNA only slightly increased the CAT activity (data not shown). These findings suggest that the difference in the activity of the S promoter observed between the two cell lines is mediated not only by titration out of the assumed repressor in the Alexander cells, but also by a positive factor. Our attempts to demonstrate the presence of such a positive element using the in viva competition assay have not been successful so far. ACKNOWLEDGMENTS We thank manuscript.

Dr. D. Shafritz and 0. Laub This research was supported

for critical reading of the by grants from MINERVA,

368 the Israel Foundation.

FAKTOR. Cancer

Association,

and

the

Israel

Cancer

DE-MEDINA,

Research

REFERENCES BERGER, I., and SHAUL, Y. (1987). Integration of hepatitis B virus: Analysis of unoccupied sites. J. Viral. 62, 1 180-l 186. CA~ANEO, R., WILL, H., HERNANDEZ, N., and SCHALLER, H. R. (1983). Signals regulating HBsAg transcription. Narure (London) 305, 336-338. CHANG, C., LIN, Y., 0-LEC, T. W., CHOU, C. K., LEE, T. S., LIU, T., PENG, F. K., CHEN, T. Y., and Hu, C. P. (1983). Induction of plasma protein secretion in a newly established human hepatoma cell line. Mol. Cell. Biol. 3, 1133-l 137. CHIRGWIN, J. M., PRZYEIYLA, A. E., MACDONALD, R. .I., and RUTTER, W. 1. (1979). Isolation of biochemically active RNA from sources enriched in ribonuclease. Biochemistry 19, 5294-5299. GORMAN, C. M., MOFFAT, L. F., and HOWARD, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2, 1044-l 051. GORMAN, C. M., RIGBY, P. W., and LANE, D. P. (1985). Negative regulation of liver enhancers in undifferentiated embryonic stem cells. Cell 42, 519-526. GOUGH, N. M., and MURRAY, K. (1982). Expression of the hepatitis B virus surface, core and E antigen genes by stable rat atid mouse cell lines. J. Mol. Biol. 162, 43-67. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467. MACNAB, G. M., ALEXANDER, J. J., LECATSA, E. M., BEY, D., and URBANOWITZ, 1. M. (1976). HBsAg produced by a human hepatoma cell line. Brir. J. Cancer 34, 509-515. MALPIECE, Y., MICHEL, M. L., CARLONI, G., REVEL, M., TIOLLAIS, P. and WEISSENEACH, J. (1983). The gene S promoter of hepatitis B virus confers constitutive expression. Nucleic Acids Res. 11, 46454654. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. MELTON, D. A., KRIEG, P. A., REBAGLIATI, M. R., MANIATIS, T., ZINN, K., and GREEN, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmides con-

AND

SHAUL

taining a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035-7056. POURCEL, C., LOUISE, A., GERVAIS, M., CHENCINER, N., DUBOIS. M. F., and TIOLLAIS, P. (1982). Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA. 1. Viral. 42, 100-l 05. SHAUL, Y., BEN-LEVY, R., and DE-MEDINA, T. (1986). High affinity binding site for nuclear factor I next to the hepatitis B virus S gene promoter. EMBO 1. 5, 1967-l 971. SHAUL, Y., RUITER, W. J., and L&US, 0. (1985). A human hepatitis B viral enhancer element. EMBO J. 4, 427-430. SHAUL, Y., ZIEMER, M., GARCIA, PH. D., CRAWFORD, R., Hsu, H., VALENZUELA, P., and RULER, W. J. (1984). Cloning and analysis of integrated HBV sequences from a human hepatoma cell line. J.

Viral. 51, 776-787. STANDRING, D. N., RUTTER, W. J., VARMUS, H. E., and GANEM, D. (1984). Transcription of the HBsAg gene in cultured murine cells initiates within the presurface region. 1. Viral. 50, 563-571. T.IOLLAIS, P., POURCEL. C., and DEJEAN, A. (1985). virus. Nature (London) 317, 489-495. TREININ, M., and LAuB, 0. (1987). Identification ment located upstream from the hepatitis Cell. Biol., in press.

The

hepatitis

of a promoter B virus X gene.

B ele-

Mol.

TREISMAN, R., GREEN, M., and MANIATIS, T. (1983). cis and trans-activation of globin gene transcription in transient assays. Proc. Nat/.

Acad. Sci. USA 80,6428-7432. VALENZUEIA, P., QUIROGA, N. ZALDIVAR, J., GRAY, P., and RUTTER, W. 1. (1980). The nucleotide sequence of the HBV genome and the identification of the major viral genes. In “Animal Virus Genetics” (B. Fields, R. Joenisch and C. Fox, Eds.), pp. 57-60. Academic Press, New York. WALKER, M. D., EDLUND, T., BOULET, A. M., and RUT~ER, W. 1. (1983). Cell-specific expression controlled by the 5’ flanking region of insulin and chymotrypsin genes. Nature (London) 306, 557-561. ZIEMER, M., GARCIA, P., SHAUL, Y., and RULER, W. J. (1985). Sequence of hepatitis B virus DNA incorporated into the genome of human hepatoma cell line. J. Viral. 53, 885-892. ZINN, K., DIMAIO, D., and MANIATIS, T. (1983). Identification of two distinct regulatory regions adjacent to the human /3 interferon gene. Cell 34, 865-879.