Differential Regulation of the Hepatitis B Virus Surface Gene Promoters by a Second Viral Enhancer*

The hepatitis B virus surface gene is transcribed from two promoters, and the resulting mRNA species code for three distinct forms of the surface antigen. We show here that the viral transcriptional trans-activator, X protein, has no effect on either promoter. However, a cis-acting element in the downstream half of the X gene, distinct from the previously mapped viral enhancer, selectively activates the major surface gene promoter. Nuclease protection and gel-shift assays reveal that multiple cellular factors bind to two sites within this DNA fragment, both of which are necessary for enhancer activity. Since this region of the viral genome is frequently deleted upon integration into the hsot chromosome in chronic hepatitis B, loss of this second enhancer can alter the relative amounts of the three forms of the surface antigen in infected hepatocytes and thus possibly contribute to cellular damage.

The hepatitis B virus surface gene is transcribed from two promoters, and the resulting mRNA species code for three distinct forms of the surface antigen. We show here that the viral transcriptional trans-activator, X protein, has no effect on either promoter. However, a cis-acting element in the downstream half of the X gene, distinct from the previously mapped viral enhancer, selectively activates the major surface gene promoter.
Nuclease protection and gel-shift assays reveal that multiple cellular factors bind to two sites within this DNA fragment, both of which are necessary for enhancer activity.
Since this region of the viral genome is frequently deleted upon integration into the hsot chromosome in chronic hepatitis B, loss of this second enhancer can alter the relative amounts of the three forms of the surface antigen in infected hepatocytes and thus possibly contribute to cellular damage.
The hepatitis B virus (HBV)' surface gene is transcribed from two tandem promoters (see Fig. lA). The upstream pre-Sl promoter transcribes an RNA species that mainly encodes the large surface antigen, while the much stronger S promoter transcribes the mRNA for the middle and small (major) surface antigens by virtue of heterogenous start sites that span the initiating AUG of the middle surface antigen (reviewed in Ref. 1). All three forms of the surface antigens, which are co-linear in the carboxyl-terminal portion, are needed for virion production, but overproduction of the large form can result in abnormal particle formation that becomes inspissated in the endoplasmic reticulum and damages the host cell (2,3). Previous studies have shown that a viral enhancer located between the surface and X genes up-regulates both promoters (4)(5)(6) ments to determine if pX also regulates the pre-Sl or S promoters.
Unexpectedly, pX has no trans-effect on the surface gene promoters.
Rather, the downstream half of the X gene is a cis-acting element that selectively enhances transcription from the S promoter.

MATERIALS AND METHODS
Plasmids-The 2.8-kilobase pair BglII fragment of HBV DNA strain adw (9), comprising the surface and X genes (see Fig. lA

AND DISCUSSION
To determine if pX trans-activated either the preS1 or S promoter, we measured the effect of deleting most of the X gene (from the NcoI to StuI sites) on transcription from these promoters.
Primer extension analysis of RNA isolated from transiently transfected hepatoma cells showed that this deletion indeed produced a significant (>&fold) drop in the amount of the S transcripts, without a significant change in the pre-Sl transcript, as judged by autoradiography of three independent transfections (Fig. 1B). However, neither a frameshift mutation in the X gene nor deletion of the upstream portion of the X gene (from NcoI to RsrII sites) had a significant effect on either promoter (Fig. 1B). These results imply that the effect of deleting the X gene resulted from loss of a cis-acting element in the downstream portion of the gene, rather than from loss of trans-activation by the X protein function. To confirm our inference, we placed a fragment of the X gene (from ApaLl to AluI sites) downstream of the CAT reporter gene in a plasmid driven by either the S or pre-Sl promoter. As predicted, this DNA fragment in either orientation was able to enhance expression from the S promoter by %-fold but had no significant effect on the pre-Sl promoter (Fig. 2). In addition, when an expression plasmid containing the X gene was co-transfected with CAT reporter genes driven by either the S promoter or pre-Sl B __)- A, the lower strand of the X gene was labeled at the 3' end and subjected to DNase I protection assay. The Roman numerals I and II indicate the two protected regions, and the arrows indicate the direct and inverted repeats present in these regions. *, a hypersensitive site; G+A, cleavage reaction at G and A residues; BSA, DNase I digestion in the presence of bovine serum albumin; Hep, digestion in the presence of hepatoma nuclear extracts. B, sequence of protected regions. The Arabic numbers indicate nucleotide position relative to the unique EcoRI site in HBV DNA (9).
( Fig. 2), even though deletion of this fragment from the HBV One or two copies of a smaller fragment of the X gene, genome resulted in a loss of enhancing activity (Fig. 1B). extending from the RsrII to StuI sites, did not have significant Therefore, it is likely that there are at least two sites within enhancing activity when placed downstream of the S promoter the X gene, one on each side of the StuI site, and that each is needed for viral morphogenesis.
On the other hand, an increased ratio of large to the middle and small forms leads to formation of abnormal surface antigen particles and loss of secretion of all three forms of the surface antigen (2,3,22,23), and the accumulated particles can become inspissated in the endoplasmic reticulum and be toxic to the host cell (2,3). Therefore, the relative strengths of the pre-Sl promoter (which encodes mostly large surface antigen) and the S promoter (which encodes the middle and small surface antigens) must be stringently controlled.
The downstream portion of the X gene, including enhancer II, is frequently deleted when HBV DNA integrates into the host chromosome during chronic infection, while the entire surface gene is usually intact in the integrated state (24)(25)(26). According to our results, this deletion would lead to an increased ratio of large to middle and small surface antigens synthesized and thus may contribute to the formation of hepatocytes with inspissated surface antigen particles ("ground glass cells") frequently seen in liver biopsy specimens of patients with chronic hepatitis B (27). Further experiments with transgenic mice will be needed to confirm this scenario for the pathogenesis of these ground glass cells, which may be important for liver injury and even carcinogenesis (3).
necessary but not sufficient for enhancer activity. To pinpoint these sites, we performed DNase I protection analysis of this fragment of DNA with nuclear extracts from hepatoma cells. Two major footprints were observed (Fig. 3A), one on each side of the StuI site. The downstream footprint (site I) covers two direct repeats of the sequence GGGAGGAG, while the upstream one (site II) is centered on an overlapping pair of palindromic sequences (Fig. 3B). Additional weaker footprints are possibly present between the two sites and upstream of site II (Fig. 3A).
Gel-shift analysis confirms that both sites I and II bind nuclear factors. Specifically, three shifted bands were observed when an oligonucleotide containing site I was incubated with HUH-~ nuclear extracts, and both were competed by unlabeled excess oligonucleotide (Fig. 4A), while two shifted bands were observed with an oligonucleotide containing site II (Fig. 4B). Additional studies will be done to determine the factors that render the pre-Sl and S promoters differentially responsive to this enhancer, since this may provide a paradigm for selective regulation of closely juxtaposed cellular promoters by enhancers.
Recently, others have shown that the portion of the X gene downstream of the StuI site (site I) enhances transcription from the HBV core and thymidine kinase promoters (20), while the portion of the X gene upstream of the StuI site (site II) enhances transcription from the SV40 early promoter (21, 28). Our results show that these two regions are interdependent in enhancing the S promoter and should be considered a single functional cis-acting unit. Therefore, the downstream half of the X gene comprises HBV enhancer II to be distinguished from the previously mapped enhancer I located between the surface and X genes (4).
Our results show that enhancer II affects the relative amounts of the different surface antigens. The balance in the synthesis of the three forms of the surface antigen is important for the HBV life cycle. On one hand, all three forms are