INTRODUCTION

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The 16.5-kDa X protein, pX, encoded by the hepatitis B virus (HBV), is expressed during viral infection (17, 28) and is implicated in HBV-mediated hepatocarcinogenesis by an unknown mechanism. In addition to its role in the viral life cycle, pX exhibits several reported activities affecting cellular transcription (35), cell growth (4, 20), and apoptotic cell death (19, 40). Importantly, the transcriptional role of pX is implicated as a mechanism by which pX deregulates cellular gene expression, resulting in hepatocyte transformation (4, 41).

Although pX does not directly bind double-stranded DNA, pX activates transcription from diverse cis-acting elements, including the AP-1 (18, 31, 36, 42), AP-2 (36), NF-B (23, 25, 37, 39, 43), and CRE sites (3, 26, 44). This transcriptional promiscuity of pX is attributed to its dual mechanism of transcriptional activation. Specifically, pX activates the mitogenic ras-raf-MAPK (5, 11) and JNK (6) pathways in the cytoplasm, leading to the activation of transcription from AP-1 and NF-B sites. pX also interacts directly with specific components of the basal transcription apparatus, including the RPB5 subunit of RNA polymerase II (7, 24), TFIIB (15, 16, 24), and TFIIH (15, 33), and with cellular bZip transcription factors (3, 26, 44). Importantly, distinct regions of pX, namely, amino acids (aa) 51 to 136 and 102 to 136, are required for the interaction of pX with RPB5 and TFIIB, respectively (24). In addition, overexpression of RPB5 in the presence of pX stimulates expression of pX-responsive promoters, supporting the idea that pX transactivation occurs via direct interaction with components of the basal transcriptional apparatus (7). Likewise, pX rescues the transcriptional inhibitory effect of the dominant-negative TFIIB mutant C34,37S, a mutant which maintains its interacting potential with pX (15). Accordingly, these data favor the model that concurrent interactions occur between pX and several components of the basal transcriptional apparatus to effect pX-mediated transactivation. However, it is not yet clear whether the direct interaction of pX with the basal transcriptional apparatus effects a specific or general enhancement of gene expression. In order for pX to effect increased transcription from specific cis-acting elements, one has to envision the recruitment by pX of the basal transcriptional apparatus to those cis-acting elements via its interaction with sequence-specific cellular transcription factors. The only known class of cellular transcription factors which interacts directly with pX and displays functional interactions in vitro and in vivo is the bZip family of transcription factors (3, 26, 44). Our previous studies (3, 44) demonstrated that the direct interaction of pX with CREB/bZip family members increases transcription in a specific manner from CRE and C/EBP binding sites. Importantly, the interaction of pX with the bZip transcription factor C/EBP was shown to synergistically activate the HBV enhancer II/pregenomic promoter (8), supporting the idea that the interaction of pX with bZip transcription factors is relevant for the expression of the viral genome.

Our previous studies (3, 44) have shown that pX interacts directly with CREB via the bZip domain, increases the affinity of CREB for either cellular or viral CRE sites by 1 order of magnitude, and increases its transcriptional efficacy in vivo in the presence of cyclic AMP. Likewise, pX interacts directly and increases the DNA-binding potential and transcriptional efficacy of inducible bZip transcription factors (C/EBP, ATF3, and ICERII), activators or repressors of transcription, all of which play important roles in hepatocyte physiology (3). However, the increased DNA-binding potential of bZip proteins (3, 44) does not explain how pX effects their increased transcriptional efficacy.

To further understand the mechanism of bZip-pX interactions and the mechanism mediating their increased transcriptional efficacy by interaction with pX, we report here, first, the delineation of a pX region required for direct CREB/bZip binding in vitro and, second, the differential requirement of pX regions in effecting bZip-mediated transactivation versus transrepression.

	MATERIALS AND METHODS

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Construction of pX deletion mutants. GST-X_79-154, GST-X_49-140, GST- X_49-115, and GST-X_49-90 were constructed as protein fusions with glutathione S-transferase (GST) by PCR. The respective pX DNA fragments were cloned into the EcoRI-HindIII sites of the pGEX-KG plasmid (14). GST-X_1-154 and GST-X_49-154 were cloned as previously described (3). The GST-X fusion proteins were expressed in Escherichia coli and purified on glutathione-Sepharose 4B resin (3). The protein concentration of the GST-X fusions was estimated by densitometric analysis by using the OPTIMAS 6.1 program and employing as a standard known amounts of bovine serum albumin (BSA), similarly analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis. In the in vitro analyses, equivalent amounts of the GST-X deletion proteins were used.

Protein-protein interaction assays. In vitro protein-protein interaction assays of pX deletion mutants in fusion with GST were carried out as previously described (3) by using recombinant, CRE-affinity-purified CREB (47), ³²P radiolabeled at the PKA (Ser₁₃₃) site (9), or in vitro-translated ³⁵S-radiolabeled ATF3 and ICERII, prepared as described earlier (3).

In vitro DNA-protein binding assays. DNA-protein binding assays were carried out by employing the somatostatin CRE as the radiolabeled probe (1, 3) and recombinant CREB (9).

Transient transfections. AML12 cells (45) were transfected by employing the calcium phosphate coprecipitation method or the FuGENE transfection protocol (Boehringer Mannheim) in the presence of 10 µM forskolin and 100 µM 3-isobutyl-1-methylxanthine (IBMX). Transfections with the luciferase reporter were performed in triplicate in 30-mm dishes. Chloramphenicol acetyltransferase (CAT) assays and luciferase assays were performed as previously described (3, 44).

	RESULTS

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Identification of the region of pX that interacts directly with CREB in vitro. Earlier studies demonstrated that pX regions A, B, and C (Fig. 1A), which are conserved to various degrees in mammalian hepadnaviruses, are required for the pX-mediated transactivation of the simian virus 40 (SV40) promoter (2, 34) and Rous sarcoma virus long terminal repeat (RSV LTR) (21). However, considering that pX mediates transactivation of pX-responsive promoters via a dual mechanism (11), in this study we investigated whether the same pX region is required for the increased transcriptional efficacy of CREB/bZip proteins by pX. Accordingly, we constructed a series of deletion mutants of pX (Fig. 1A) by PCR-based cloning of the respective fragments into the pGEX-KG vector (14). The rationale for constructing these mutants is that pX_49-154 lacks the first 48 aa of pX known to repress pX-mediated transactivation (29) and pX_79-154 starts at an internal Met residue (22). pX_49-140 contains regions A, B, and C (Fig. 1A) required for pX-mediated transactivation of the SV40 promoter (2, 34) and RSV LTR (21). pX_49-115 contains regions A and B, and pX_49-90 contains only region A (Fig. 1A).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   (A) Diagram of constructed pX deletion mutants. pX regions A, B, and C are conserved to various degrees in hepadnaviruses and are essential for transactivation. C61 and C69 are conserved cysteines. The polymerase II, RPB5 subunit, TFIIH, and TFIIB interacting regions are indicated. (B) Coomassie blue-stained SDS gel (14%) of purified (4), recombinant GST-X and the indicated pX deletions. , GST-X fusion protein; *, endogenously cleaved GST portion. (C) Western blot of recombinant wt pX, pX49-154, pX79-154, pX49-140, pX49-115, and pX49-90 (GST fusions) detected with a polyclonal pX-specific antibody. , GST-X fusion protein. Protein bands smaller than the GST-X fusion proteins are degradation products.

8

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Kd determination of CREB-pX interaction. GST-X1-154 resin () (5 µg) was incubated with increasing amounts of 32P-CREB (0.2 to 62.5 nM) for 3 h at 4°C in buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, 0.1% Triton X-100, 5 mM dithiothreitol, 5 mM phenylmethylsulfonyl fluoride, and 50 µg of BSA per ml. The beads were washed six times and eluted by boiling in SDS-PAGE loading buffer. Analysis was done on SDS-12% PAGE gels. Equivalent amounts of GST-bound resin () or equal amounts of glutathione beads () were used as the control. Bound CREB was plotted against the total concentration of CREB. The Kd value was calculated as the concentration of CREB at which half-maximal binding to GST-X was observed. The experiment was repeated three times and shows a Kd value of approximately 1.8 × 108 M. A representative experiment from three independent experiments is shown above.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Activity of pX deletions in vitro. (A) Protein-protein interaction assay with equivalent amounts of GST-X deletion mutants (3 µg) incubated with 32P-CREB (5 to 10 ng) for 3 h at 4°C in buffer containing 25 mM HEPES (pH 7.5), 100 mM KCl, 0.1% Triton X-100, 5 mM dithiothreitol, 5 mM phenylmethylsulfonyl fluoride, and 50 µg of BSA per ml. The beads were washed six times and eluted by boiling in SDS-PAGE loading buffer. Analysis was done by SDS-PAGE on 12% gels. A total of 12 to 15 µg of GST bound to resin was used as a control. (B) Quantitation of relative CREB binding to each GST-X deletion mutant compared to wt pX carried out by scintillation counting of the gel excised bands. Nonspecific binding of CREB to the GST-resin control is subtracted from all of the plotted values. The data shown are from four independent experiments. (C) DNA-protein binding assays of recombinant CREB in the presence of pX mutants were carried out as described previously (3) with 10 ng of recombinant CREB and 10,000 cpm of radiolabeled CRE, with an equivalent amount of the following: + lanes, GST-X49-140, GST-X49-115, and GST-X49-90;  lanes, recombinant GST protein, as indicated. Lanes containing GST-X49-90 were exposed for 2 days at 80°C during autoradiography, as opposed to 18 h of exposure for the other lanes.

Identification of the minimal functional region of pX that enhances the DNA-binding potential of CREB in vitro. To determine the functional significance of the interaction of pX_49-140 and pX_49-115 with CREB, we tested the potential of these pX deletion mutants to enhance the CRE-binding potential of CREB in vitro. Gel retardation assays were performed by employing radiolabeled CRE as the probe (3) and recombinant CREB (9) in the presence or absence of the pX deletion mutants pX_49-140, pX_49-115, and pX_49-90. Addition of equivalent amounts of either pX_49-140 or pX_49-115 enhanced the CRE-binding potential of CREB (Fig. 3C). By contrast, addition of equivalent amounts of pX_49-90, which displays 80% reduced binding to CREB in vitro (Fig. 3B), failed to enhance the CRE-binding potential of CREB (Fig. 3C). Accordingly, we have delineated a minimal functional region of pX containing 67 aa residues, pX_49-115, that is required for direct CREB interaction and enhanced CREB/CRE binding in vitro.

In vivo activity of the pX mutants in AML12 hepatocytes. To confirm the in vitro results by in vivo analyses, we performed two types of assays in AML12 hepatocytes. First, we examined whether pX_49-140 and pX_49-115 interact directly with CREB in vivo by employing the mammalian two-hybrid assay. Second, we examined by transient-transfection assays in AML12 cells whether these pX deletion mutants are active in enhancing the transcriptional efficacy of CREB in vivo.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Activity of pX deletions in vivo. (A) Mammalian two-hybrid assay of pX deletion mutants in AML12 cells. A total of 500 ng of Gal4UAS-luciferase reporter was cotransfected by the FuGENE transfection protocol, with 700 ng each of RSV-CREB-VP16 and RSV-X-Gal4 expression vectors per 30-mm culture dish, performed in triplicate. Cells were harvested 18 h after transfection, and luciferase activity was quantitated and expressed per microgram of protein extract as previously described (3). Results are from three independent experiments. (B) Transient transfection of HBV pX mutants in AML12 cells. A total of 3 to 5 µg of CRE3-CAT reporter plasmid was cotransfected with pCMV-X-NLS (20 ng), pCMV-X49-140 (100 ng), and pCMV-X49-115 (50 ng) expressor plasmid. Control transfections contained equivalent amounts of pCMV-empty vector. The optimal amounts of pCMV-X expression vectors shown (wt and mutants) were established by titration analyses. Transfected cells were treated with 10 µM forskolin and 100 µM IBMX and harvested at 24 h. CAT assays were performed as described earlier (3). Relative CRE3-CAT induction values per microgram of protein extract were quantitated from three independent experiments.

Interaction of pX_49-140 and pX_49-115 with bZip repressors ATF3 and ICERII. Since pX displays promiscuity in its interaction with bZip proteins (3), we examined whether these characterized pX deletion mutants display the same requirements in interacting with various bZip proteins. We selected the bZip proteins ATF3, ICERII, and NF-IL6, which were shown previously to undergo functional interactions with pX (3). In vitro protein-protein interaction assays were carried out by using the GST fusion proteins of wt pX, pX_49-140, pX_49-115, and pX_49-90 with in vitro-translated ICERII, ATF3, and the bZip domain of NF-IL6 (data not shown). We observed (Fig. 5) that ATF3 interacts with both pX_49-140 and pX_49-115 in a way similar to wt pX. ICERII also interacts with both pX_49-140 and pX_49-115 deletions, although in comparison to wt pX it displays reduced direct binding to these regions. Like CREB, neither ATF3 nor ICERII displays appreciable direct binding to pX_49-90 (Fig. 5). Therefore, all of the bZip proteins tested interact with the same minimal region of pX, spanning aa 49 to 115, which is also required for interaction with CREB in vitro (Fig. 3) and in vivo (Fig. 4A). These results support the idea that a common mechanism exists for the interaction of bZip transcription factors with viral pX.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   Interaction of pX deletions with ATF3 and ICERII. (A) Protein-protein interaction assays of GST-X, GST-X49-140, GST-X49-115, and GST-X49-90 with in vitro-translated, [35S]methionine-radiolabeled ATF3 (3 µl) and ICERII (7 µl) as indicated. Experimental conditions were as described in Fig. 3A. (B) Quantitation of relative ATF3 and ICERII binding to each GST-X deletion mutant compared to wt pX as determined by scintillation counting of the gel-excised bands. Nonspecific binding of ATF3 and ICERII to the GST-bound resin is subtracted from all of the plotted values. The data shown are from three independent experiments.

In vivo activity of the pX mutants in mediating repression by ATF3 and ICERII. To assess the activity of deletions pX_49-115 and pX_49-140 in mediating enhanced transrepression by ATF3 and ICERII, expression vectors encoding each of the pX deletion constructs were cotransfected with ATF3 or ICERII in AML12 cells by employing the CRE-driven reporters CRE-luciferase (Fig. 6A) and CRE³-CAT (Fig. 6B), respectively. Earlier studies demonstrated that ATF3 and ICERII repress CRE³-CAT expression, and pX coexpression further increases their transrepression efficacy (3). The results shown in Fig. 6 demonstrate that, like wt pX, both pX_49-140 and pX_49-115 further promote the repression efficacy of ATF3 and ICERII. Therefore, while the pX-mediated enhancement of CREB/CRE transcription requires the pX region spanning aa 49 to 140 (Fig. 4B), the pX-mediated increase in the repression efficacy of ATF3 and ICERII occurs as efficiently with pX_49-115 (Fig. 6). Importantly, the functional activity of pX_49-115 in promoting ATF3- and ICERII-mediated repression excludes the possibility that this deletion mutant is unstable in vivo.

View larger version (15K): [in this window] [in a new window]   FIG. 6.   Transient transfection of HBV pX mutants in AML12 cells in the presence of bZip repressors ATF3 and ICERII. (A) A total of 600 ng of CRE-luciferase reporter plasmid was cotransfected by the FuGENE protocol (Boehringer Mannheim) with 100 ng of pCMV-X-NLS, pCMV-X49-140, and pCMV-X49-115 expressor plasmid in the presence of 50 ng of ATF3 expression vector. (B) CRE3-CAT reporter plasmid (5 µg) cotransfected by the calcium phosphate protocol (BRL) with pCMV-X-NLS (20 ng), pCMV-X49-140 (50 ng), and pCMV-X49-115 (50 ng) expressor plasmid in the presence of 0.5 µg of ICERII. Optimal amounts of the indicated expression vectors transfected in AML12 cells were determined by titration analyses. Relative CRE3-CAT activities per microgram of protein extract were quantitated from three independent experiments.

	DISCUSSION

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Our earlier studies have demonstrated that the interaction of pX with bZip transcription factors CREB, ICERII, ATF3, and NF-IL6 increases their DNA-binding affinity in vitro and their transcriptional efficacy in vivo (3, 44). In the present study we have employed the CREB-pX model system to examine the mechanism by which pX increases the transcriptional efficacy of bZip transcription factors. Our approach was to define a minimal, functional region of pX required for CREB interaction and activity. The rationale for undertaking these studies was that the definition of a minimal region of pX will provide convincing evidence to demonstrate the specificity of CREB (bZip)-pX interactions and will also provide a useful tool for biophysical analyses of pX and the bZip-pX complex.

We report the delineation of a 67-aa region of pX, spanning aa 49 to 115, as the minimal region required for direct CREB binding (Fig. 3 and 4A) and for the enhancement of the CRE-binding potential of CREB (Fig. 3C). Interestingly, although this minimal region of pX enhances the CRE-binding potential of CREB in vitro, it only minimally increases the transcriptional efficacy of CREB in vivo (Fig. 4B). By contrast, pX_49-140 enhances both the CRE-binding potential of CREB (Fig. 3C) and its transcriptional efficacy in vivo (Fig. 4B). The transcriptionally active deletion pX_49-140, tested by CREB/CRE-driven transcriptional induction, contains pX regions A, B, and C, all of which are required for transactivation of both the SV40 early promoter (34) and the RSV LTR (21). Importantly, this pX region has been shown to contain binding sites for both the RPB5 subunit of RNA polymerase II and TFIIB, mapped at amino acid residues 51 to 136 and 102 to 136, respectively (24). By contrast pX_49-115, which results in a minimal increase in the transcriptional efficacy of CREB (Fig. 4B), lacks region C; this region contains a portion of the binding sites for RPB5 and TFIIB (25) and nine conserved amino acids (132 to 140), which were shown to be required for pX-mediated transactivation (2, 34).

Therefore, it is interesting to speculate that concomitant interactions of pX with the bZip transcription factors and with one or more components of the basal transcriptional apparatus account for the pX-mediated enhancement of CRE transcription in vivo (Fig. 7). Since both pX_49-140 and pX_49-115 are functional in vitro (Fig. 3C), whereas only pX_49-140 is functional in vivo (Fig. 4B), these data suggest that the interaction of pX with CREB may bridge specific components of the transcriptional apparatus to effect the observed increased transcriptional efficacy of CREB.

View larger version (19K): [in this window] [in a new window]   FIG. 7.   Model depicting concomitant interactions of pX with bZip transcription factors and the basal transcriptional apparatus. (A and B) pX-mediated CREB transactivation effected by pX49-140 (containing regions A, B, and C) and pX49-115 (containing regions A and B), respectively. (C and D) pX-mediated ICERII transrepression effected by pX49-140 and pX49-115, respectively.

Based on in vitro transcription analyses and subcellular fractionation studies (30), it has been demonstrated that activated CREB interacts with the basal transcriptional apparatus at two distinct levels: (i) phospho(Ser₁₃₃)-CREB recruits RNA polymerase II via interaction with CBP and (ii) the Q₂ region of CREB associates with TFIID via interaction with hTAF_II130. Accordingly, we propose that pX_49-140, containing the conserved regions A, B, and C, tethers or stabilizes the transcriptional initiation complex via interaction with TFIIB or the RPB5 subunit of RNA polymerase II, as shown in Fig. 7A. According to our model, deletion of pX region C (Fig. 7B) only minimally promotes the interaction with the basal transcriptional apparatus, resulting in minimal induction of CREB-driven transcription. The significance of our observations and our proposed model is that CREB and the various bZip proteins (3) represent nonartificial, physiological cellular targets of pX (3, 26, 44). Furthermore, since bZip cis-acting elements are present within the HBV enhancers I and II (8, 12, 26), our model is relevant not only for cellular gene expression but also for the expression of the viral genome. Further studies are required to demonstrate directly the role of pX in bridging specific components of the basal transcriptional apparatus to CREB/bZip transcription factors.

Regarding the mechanism of the increased repression efficacy displayed by bZip repressors in the presence of pX (3), our results (Fig. 6) demonstrate that the minimal pX_49-115 is as effective as the wt pX in increasing the repression efficacy of ATF3 and ICERII in vivo. Since pX_49-115 interacts directly with bZip proteins in vitro (Fig. 3 and 5) and in vivo (Fig. 4A) and also increases the CRE-binding potential of CREB in vitro (Fig. 3C), we interpret these observations as direct evidence in support of the importance of the increased DNA-binding potential of bZip proteins by pX in mediating enhanced bZip-driven transactivation or transrepression. Moreover, these results lend further support to the importance of the interaction of pX with the basal transcriptional apparatus (7, 15, 16, 24, 33). ICERII represses CRE-mediated transcription due to lack of an activation domain (27). Accordingly, ICERII is an example of a transcriptional repressor not engaging the basal transcriptional apparatus. The increased DNA-binding potential effected by pX_49-115 is sufficient to mediate repression by ICERII (Fig. 7C and D). We conclude that while pX-dependent coactivation is crucial in mediating CREB/CRE-driven transactivation, the interaction of pX with the basal transcriptional apparatus is not required for ICERII- or ATF3-driven transcriptional repression (Fig. 6).

Since the minimal, functional pX region required for CREB interaction is also required for direct interaction with other bZip proteins (ATF3, ICERII, and NF-IL6), this observation (Fig. 5) supports the idea that a common mechanism exists for the interaction of pX with bZip family members. Accordingly, this minimal region of pX is a powerful tool for biophysical studies, in order to determine the structural features of the CREB (bZip) interacting region of pX and its interaction with CREB/bZip family members. Importantly, no structural information is yet available for pX. Both pX mutants, spanning amino acid residues 49 to 140 and 49 to 115, express 1 to 2 mg of soluble protein per liter of bacterial culture (data not shown). Thus, these pX deletion mutants are amenable to biophysical analyses. The information derived from the biophysical studies should provide an important contribution toward understanding pX and, by examining naturally occurring pX variants, will provide a new approach to address the clinical relevance of CREB (bZip)-pX interactions. Very little is known of the sequence variations of pX, mainly because the pX gene overlaps the viral polymerase gene (46) and the precore gene (32) and carries several signals critical to the replicative viral lifecycle (13). It will be interesting to examine these naturally occurring sequence variations from the perspective of CREB-bZip interactions, employing our well-defined in vitro and in vivo functional assays (3, 41, 44).