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Originally published In Press as doi:10.1074/jbc.M910378199 on March 15, 2000
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J Biol Chem, Vol. 275, Issue 20, 15157-15165, May 19, 2000


Structural and Functional Characterization of Interaction between Hepatitis B Virus X Protein and the Proteasome Complex*

Zhensheng Zhang, Nobuyuki Torii, Akihiro Furusaka, Navara Malayaman, Zongyi Hu, and T. Jake LiangDagger

From the Liver Diseases Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for a 16.5-kDa protein known as hepatitis B virus X protein (HBX). The importance of HBX in the life cycle of HBV has been well established, but the underlying molecular function of HBX remains controversial. We previously identified a proteasome subunit PSMA7 that interacts specifically with HBX in the Saccharomyces cerevisiae two-hybrid system. Here we demonstrate that PSMC1, an ATPase-like subunit of the 19 S proteasome component, also interacts with HBX and PSMA7. Analysis of the interacting domains among PSMA7, PSMC1, and HBX by deletion and site-directed mutagenesis suggested a mutually competitive structural relationship among these polypeptides. The competitive nature of these interactions is further demonstrated using a modified yeast two-hybrid dissociator system. The crucial HBX sequences involved in interaction with PSMA7 and PSMC1 are important for its function as a transcriptional coactivator. HBX, while functioning as a coactivator of AP-1 and acidic activator VP-16 in mammalian cells, had no effect on the transactivation function of their functional orthologs GCN4 and Gal4 in yeast. Overexpression of PSMC1 seemed to suppress the expression of various reporters in mammalian cells; this effect, however, was overcome by coexpression of HBX. In addition, HBX expression inhibited the cellular turnover of c-Jun and ubiquitin-Arg-beta -galactosidase, two well known substrates of the ubiquitin-proteasome pathway. Thus, interaction of HBX with the proteasome complex in metazoan cells may underlie the functional basis of proteasome as a cellular target of HBX.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human hepatitis B virus (HBV)1 belongs to a group of hepadnaviruses that includes the hepatitis viruses of the woodchuck, ground squirrel, tree squirrel, Pekin duck, and heron. HBV has a unique fourth open reading frame, termed the hepatitis B virus X (HBX) gene. HBX gene is well conserved among the mammalian hepadnaviruses and codes for a 16.5-kDa protein (1, 2). The protein can activate the transcription of a variety of viral and cellular genes (3-5) and induce liver cancer in certain transgenic mouse model (6). Since HBX does not bind to DNA directly, its activity is thought to be mediated via protein-protein interactions. HBX has been shown to enhance transcription through AP-1 and AP-2 (7-9) and to activate various signal transduction pathways (10, 11). Several recent studies have also identified possible cellular targets of HBX, including members of the CREB/ATF family (12, 13), the TATA-binding protein (14), RNA polymerase subunit RPB5 (15, 16), the UV-damaged DNA-binding protein (17), and the replicative senescence p55sen (18). HBX has also been shown to interact with p53 and inhibit its function (19, 20). Furthermore, HBX possesses amino acid sequence homology to the functionally essential domains of Kunitz-type serine proteases inhibitors and mutation of this putative motif inactivates the transactivation function of HBX (21).

Using the Saccharomyces cerevisiae two-hybrid system (22, 23), we previously identified an alpha  proteasome subunit, PSMA7, as a putative cellular target of HBX. We demonstrated that this interaction may be functionally important in the pleiotropic effect of HBX (24). In the present study, we identified another HBX-interacting clone as the proteasome subunit PSMC1, which is an ATPase-like member of the 19 S regulatory factor (25-27). The interacting domains of PSMA7, PSMC1, and HBX were characterized, and the specificity of these interactions was further evaluated using a modified yeast two-hybrid dissociator system. We also studied the transactivation function of HBX in mammalian cells and yeasts, and further demonstrated the functional importance of HBX-PSMC1 interaction.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains-- Four S. cerevisiae strains were used, and their genetic backgrounds are summarized in Table I. EGY48 was used in the standard yeast two-hybrid system, and EGY40 in the modified yeast two-hybrid dissociator system. KNY14 (inactivated GCN4 gene) and KNY24 (wild-type GCN4) (28, 29) were used to study the effect of HBX on GCN4, the yeast AP-1 ortholog. The MaV103 yeast strain was used to test the effect of HBX on the Gal4 transactivator.

                              
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Table I
Genetic background of S. cerevisiae strains

Plasmid Construction-- For the yeast two-hybrid system, the HBX gene was fused to the lexA DNA binding domain of pEG202 as pEG202HBX (24). The reporter construct was lexAop-lacZ gene, which permits determination of interaction based on beta -galactosidase (beta -gal) activity. Two lacZ reporter constructs, pSH18-34 and JK103 (both with URA3 marker), contained eight and two LexA binding sites, respectively (23). pRF4-6NL, a TRP1 plasmid containing Gal1 promoter that is galactose-inducible, was used for expression of the "dissociator" in the modified yeast two-hybrid dissociator system (30). pCL1, a LEU2 plasmid expressing GAL4, and p2.5 plasmid containing the HIS3 marker have been described (22, 31). p2.5HBX was generated by inserting HBX fragment (EcoRI-NotI) from pEG202HBX into p2.5 plasmid. Mutations in HBX were introduced by PCR-based mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA). The HBX mutants have been described previously (24) and HBXmd and HBXsm, containing the middle and small HBX, respectively, were generated by PCR. Deletion mutants of PSMA7 and PSMC1 were generated using convenient restriction sites or by PCR (for map, see Fig. 2). Chimeric constructs of yeast and human PSMA7 constructs (YHA7.169, YHA7.199, and YHA7.227) were generated by PCR, exchanging corresponding regions of yeast with human sequences. All mutant constructs were confirmed by DNA sequencing. pYepHBX was generated by ligation of the ADH1 promoter (PstI-HindIII fragment from pJG7-1) (31) and a HindIII-BamHI fragment from JG4-6HBX containing HBX and ADH1 transcription terminator into the PstI and BamHI sites of a LEU2 plasmid pYepLac181 (provided by Alan Hinnebusch, NICHD, National Institutes of Health, Bethesda, MD). Plasmid B2079 (TRP1 marker) (28), containing a GCN4-responsive lacZ reporter, was also provided by Alan Hinnebusch. Gal4 reporter gene p17X4TATA-CAT, which contains four copies of Gal4 binding sites, the thymidine kinase minimal promoter and the chloramphenicol acetyltransferase (CAT) reporter; and plasmid pCEP4GLVP which contains a fusion of Gal4 DNA binding domain and the VP-16 acidic activation domain driven by the SV40 promoter, were provided by Sophia Tsai (Baylor College of Medicine, Houston, TX). and Robert Kingston (Massachusetts General Hospital, Boston, MA), respectively. The CMV-HA-c-Jun containing the full-length c-Jun tagged with the influenza virus hemagglutinin (HA) epitope at its N terminus was provided by Mathias Treier (European Molecular Biology Laboratory, Heidelberg, Germany) (32). The CMV-Arg-beta -gal plasmid was generated by inserting the DNA fragment coding for ubiquitin-Arg-beta -galactosidase (provided by Alexander Varshavsky, California Institute of Technology, Pasadena, CA) (33) into the plasmid pCDNA1.

Protein Analysis-- For in vitro binding experiment, [35S]Met-labeled HBX and PSMA7 proteins were generated using Rabbit Reticulocyte Extract system (Promega, Madison, WI). PSMC1 (full-length) and PSMA7 (aa 137-248) were cloned into the pGEX-KG vector (Amersham Pharmacia Biotech) and expressed as a glutathione S-transferase (GST) fusion protein, which were purified by glutathione-coupled agarose beads. The translated proteins were then incubated with the protein-bound beads in NETN buffer (20 mM Tris, pH 8.0, EDTA 1 mM, 100 mM NaCl, 0.5% Nonidet P-40) at room temperature for 1 h with constant mixing. The beads were washed extensively with the same buffer and the bound proteins were subjected to 15% SDS-PAGE and PhosphorImager (Storm, Molecular Dynamics, Sunnyvale, CA) analysis. For the pulse-chase experiment, cells were lysed directly in a 10-cm dish with 1 ml of cold standard lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF. The lysed cells were centrifuged at 13,000 × g for 15 min at 4 °C to remove the nuclei and other insoluble cell debris. Immunoprecipitation was performed by incubating the cell lysates with antibody first and then protein G-coupled Sepharose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunoprecipitates were separated by SDS-PAGE and analyzed with a PhosphorImager. The monoclonal antibody 12CA5 specific for the HA epitope and the anti-beta -gal monoclonal antibody were purchased from Roche Molecular Biochemicals.

Yeast Transformation and beta -Galactosidase Assay-- Standard yeast two-hybrid system was performed as described (23). For the modified yeast two-hybrid dissociator system, four pairs of interacting proteins, HBX-PSMA7, PSMC1.1-PSMA7, Max-Mxi1 (34), and PreS2-B312 were cloned into the LexA DNA binding domain vector (pEG202: ADH1 promoter, HIS3 marker) and the B42 acidic activator vector (pRF25: ADH1 promoter, TRP1 marker) (30), respectively. Yeast strain EGY40 was first transformed with these interacting pairs and then the lacZ reporter plasmid JK103. Positive interacting clones were then retransformed with the dissociator construct containing the dissociator gene cloned into the pRF4-6NL vector (Gal1 promoter, LEU2 marker). The yeast transformants were grown on plates containing Glu/CSM-His-Leu-Trp-Ura (Bio 101, Inc., Vista, CA) at 30 °C for 2-3 days until colonies were visible. 10 independent colonies from each group were streaked onto the Gal/CSM-His-Leu-Trp-Ura plates (galactose to induce the expression of the dissociator) and incubated at 30 °C for 2-3 days. The streaked colonies were then harvested, lysed with acid-washed 425-600-µm glass beads (Sigma), and assayed for beta -gal activities using the Galacto-Light kit (Tropix, Bedford, MA).

DNA Transfection and Transactivation Assay-- Three reporter plasmids were used for HBX transactivation assays in the HepG2 cells. The RSV-Luc, the AP-1-CAT (containing four AP-1 sites adjacent to a minimal human metallothionein IIA promoter), and the mTK-Luc (containing minimal thymidine kinase promoter) have been described previously (24). The human hepatoma HepG2 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum in a humidified incubator (5% CO2). Transient transfection of HepG2 cells in a 35-mm well was carried out using the DNA transfection kit (5 Prime right-arrow 3 Prime, Inc., Boulder, CO). Luciferase assay was performed with a Monolight luminometer (Analytical Luminescence Laboratory, San Diego, CA). CAT assay was performed using the CAT enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction of HBX with Proteasome Subunits PSMA7 and PSMC1-- Using the yeast two-hybrid system, we previously identified several independent clones that interacted specifically with HBX (24). One strongly interacting clone was identified as an alpha  subunit of the proteasome complex, PSMA7 (24). One of the other clones, PSMC1, was an ATPase-like subunit of the 19 S regulatory component of the 26 S proteasome complex (25, 26). In addition, the PSMA7 clone also interacted specifically with the PSMC1 clone in the yeast two-hybrid system.

To demonstrate the interaction of HBX and the proteasome subunits in vitro, we constructed two GST fusion expression plasmids, one with PSMA7 and the other with PSMC1. The GST fusion proteins expressed in bacteria were purified with glutathione beads and incubated with the in vitro translated, [35S]Met-labeled HBX or PSMA7 proteins. The beads were then washed and the bound polypeptides subjected to SDS-PAGE analysis (Fig. 1). HBX bound specifically to GST-PSMA7 and -PSMC1 but not GST. Similar binding was also shown between PSMA7 and GST-PSMC1 or -HBX. The binding appeared to be stronger between PSMA7 and PSMC1 than that between HBX and PSMA7. This finding is consistent with the relative strength of interactions among these proteins in the yeast two-hybrid system. In addition, these binding results corroborated the interaction of these polypeptides in the yeast two-hybrid system.


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  		Fig. 1.   In vitro binding of HBX with PSMC1 and PSMA7. In vitro translated, [35S]Met-labeled PSMC7 and HBX were incubated with GST, GST-HBX, or GST-PSMC1 (lanes 2-4), and GST, GST-PSMA7 or GST-PSMC1 (lanes 6-8) bound to the glutathionine beads, respectively. The same quantities of GST proteins are used in each binding assay. The bound proteins were washed extensively, denatured, electrophoresed, and exposed to a PhosphorImager. The in vitro translated proteins (about one-tenth of what was used for the binding) are shown in lane 1 as PSMA7 and in lane 5 as HBX. The other lanes are as indicated. The molecular mass markers are shown on the left.

To characterize the interacting domains of HBX, PSMC1 and PSMA7, various deletion and site-directed mutants of each cDNA were generated and tested for their interactions in the yeast two-hybrid system. In our previous study, we determined that the C-terminal region (aa 137-248) of PSMA7 is important for binding to HBX (24). To further characterize the sequences of PSMA7 important for binding to HBX and PSMC1, we generated additional deletion mutants and tested their interactions with HBX and PSMC1 (Fig. 2A). The deletion mutants H7.198 (aa 137-198), H7.227 (aa 137-227), and H7.230 (aa 137-230) exhibited no binding to either HBX or PSMC1, while H7.237 (aa 137-237) retained nearly full binding activity to HBX and PSMC1 (Fig. 2A). In addition, the N-terminally truncated construct H7.169 (aa 169-248) had binding activity similar to that of H7.237 (Fig. 2A). Since the yeast PSMA7 ortholog (YPSM7) (35) has regions of sequence homology with the PSMA7, its interaction with HBX and PSMC1 was analyzed (Fig. 2A). The YPSMA7 demonstrated no binding to HBX but had a weak binding to PSMC1 (Fig. 2A), suggesting that human PSMA7 exhibits selective binding to HBX and human PSMC1. To further characterize the binding domain between HBX, PSMC1, and PSMA7, we generated three constructs that are chimeric for human and yeast PSMA7: YH7.169, YH7.199, and YH7.227. YH7.169 and YH7.199 had full binding activity to HBX, while YH7.227 demonstrated no binding to either (Fig. 2A). On the other hand, YH7.199 interacted with PSMC1 as well as the HY7.169 did, but its binding to HBX was weaker than that to YH7.169. Taken together, aa 199-237 of the human PSMA7 contains the necessary structural information for binding to both HBX and PSMC1. However, the observed minor difference in the mapping results suggested that the binding domains of PSMA7 with HBX and PSMC1 may be slightly different. Comparison of human and yeast PSMA7 sequences shows that major sequence divergence in this region probably accounts for the difference in binding.


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  		Fig. 2.   Characterization of the interacting domains among HBX, PSMA7, and PSMC1. A, binding of PSMA7 to HBX and PSMC1. The N-terminally truncated form of human PSMA7 (aa 137-248), which has full binding activities to HBX and PSMC1, was used for generation of all mutant constructs. The various human and chimeric yeast-human PSMA7 constructs were analyzed for interaction with HBX or PSMC1 (the construct PSMC1.1 was used) in the yeast two-hybrid system. The clear area represents the human sequences and the shaded area the yeast sequences. The numbers above indicate the amino acid positions of the proteins. B, binding of PSMC1 to PSMA7 and HBX. The full-length human and yeast PSMC1s, as well as various deletion mutants, were constructed and analyzed for interaction with PSMA7 and HBX. C, binding of HBX to PSMA7 and PSMC1. HBX mutants were generated as described. The transactivation activity and binding strength to PSMA7 and PSMC1.1 of each HBX construct are shown. For each two-hybrid analysis, the deletions and mutants of the first construct were fused to the LexA DNA binding domain of pEG202 and the two interactors fused to the B42 activation domain of JG4-5. The binding strength of each interacting pair was determined by beta -galactosidase activity in the yeast two-hybrid system. The lacZ reporter pSH18-34 was used in these experiments. For each experiment, 10 yeast tranformants were analyzed for each pair of interactors and the results (means ± standard deviations) are representative of at least three separate experiments. CTRL, control.

Fig. 2B showed that the full-length PSMC1 (aa 1-440) interacted with both PSMA7 and HBX. The PSMC1.1 construct (aa 123-440) containing N-terminally truncated PSMC1, also interacted with PSMA7 and HBX, although the interaction with HBX was weaker than that of the full-length PSMC1. On the other hand, the strength of binding to PSMA7 appeared to be much higher for the truncated than the full-length PSMC1. Two additional deletions, PSMC1.2 (aa 123-316) and PSMC1.3 (aa 317-440), demonstrated no binding to HBX. In contrast, PSMA7 retained high binding activity to PSMC1.3 but not to PSMC1.2. Collectively, these results suggested that PSMC1 interacts with PSMA7 and HBX via distinct domains: a N-terminal domain to HBX and a C-terminal domain to PSMA7. Two constructs of the yeast PSMC1 (36) were assayed for interaction with the HBX and PSMA7. The yeast PSMC1 interacted weakly with HBX, but exhibited moderate binding to the human PSMA7. Similar to the human pairs, the yeast PSMC1 interacted well with the yeast PSMA7 (data not shown). Sequence divergence between the human and yeast PSMC1 subunits may again account for the difference in binding to HBX.

The Second Kunitz-type Domain of HBX Is Essential for Interaction with Proteasome Subunits PSMA7 and PSMC1-- Functional mapping of HBX has defined two structural domains that are crucial for the transactivation function of HBX (21, 24, 37). These two domains appear to overlap with the putative Kunitz-type domain of protease inhibitor that is present in both HBX and WHVX (21, 24). Our previous mutagenesis studies established a structural and functional association of mutations in the second Kunitz-type domain of HBX with respect to interaction between HBX and PSMA7; HBX mutants defective in binding to PSMA7 were also negative for transactivation (24). To determine the interacting domain of HBX with PSMC1, we carried out similar experiments using various HBX mutants (Fig. 2C). The results showed that the first Kunitz-type domain is also not important for binding to PSMC1. Analysis of constructs with mutations in the second Kunitz-type domain revealed that H139D mutation abolished binding of HBX to both PSMA7 and PSMC1, while C137S and R138Q mutations had no effect on binding to PSMA7 but reduced markedly the binding of HBX to PSMC1 (Fig. 2C). Together with our previous data on PSMA7 and HBX interaction (24), the current study suggests that the second Kunitz-type domain of HBX is essential for binding to both proteasome subunits PSMA7 and PSMC1, albeit the binding motifs may not be exactly the same.

HBX gene, through alternative translation initiations, could potentially encode three HBX polypeptides that may function differentially to transactivate polymerase II and III promoters (38). From the data above, we reason that all three forms of HBX should bind equally well to PSMA7 and PSMC1. Two HBX constructs, HBXmd and HBXsm, containing HBX sequences from the second and third in-frame start codons, respectively, were generated. Interactions of all three forms of HBX with either PSMA7 or PSMC1 were studied using the yeast two-hybrid system. The results showed that both HBXmd and HBXsm bound equally well to PSMA7 and PSMC1 as the full-length HBX, despite both constructs scoring negative for transactivation using RSV-Luc as the reporter gene (Fig. 2C). Taken together, these results suggested that the second Kunitz-type domain of HBX is essential for interaction with the proteasome subunits. The N terminus of HBX, which includes the first Kunitz-type domain, is not necessary for binding to proteasome subunits, but may interact with other cellular factor(s) that is equally important for the function of HBX. Recent reports have suggested that the N-terminal domain of HBX interacts with a DNA repair enzyme UVDDB (39), the RNA polymerase subunit RPB5 (15, 16), and the general transcriptional factor TFIIH (14, 40).

The Two-hybrid Dissociator System Defines a Competitive Interaction among HBX, PSMA7, and PSMC1-- To further define the structural relationship of interactions among HBX and the proteasome subunits PSMA7 and PSMC1, we adopted a modified yeast two-hybrid dissociator system (30). The system is diagrammatically summarized in Fig. 3A. Briefly, two potentially interacting protein partners (bait and prey) were expressed in the standard two-hybrid system. The third construct expressing the dissociator protein under the Gal1 promoter (pRF4-6) was then introduced and the beta -gal activity (lacZ reporter) analyzed after induction of the dissociator by galactose. If the dissociator interacts with either the bait or prey through similar structural domains, a competitive inhibition of the original interaction would result, leading to a decreased beta -gal activity. In the present study, four groups of interactors were evaluated using this system (Fig. 3B). Two interacting pairs from this study, HBX-PSMA7 and PSMC1.1-PSMA7, and two unrelated interacting pairs, Max-Mxi1 (34) and PreS2-B31, as controls were studied. PSMC1.1 was used instead of the full-length PSMC1 because it conferred a stronger binding to PSMA7. Since previous mapping results revealed that both HBX and PSMC1 interacted with PSMA7 through the PSMA7 C-terminal domain, we expected that the interaction between HBX and PSMA7 should be competed by either HBX or PSMC1.1. As predicted, HBX-PSMA7 interaction was inhibited by HBX (to 71% of the activity) and more significantly by PSMC1.1 (to 55%) (Fig. 3B). Similarly, the PSMC1.1-PSMA7 interaction was dramatically reduced by PSMC1.1 (to 47% activity) but only modestly by HBX (to 75%). These results are consistent with the relatively stronger interaction of PSMC1.1-PSMA7 as compared with that of HBX-PSMA7 interaction (about 30% of PSMC1.1-PSMA7 interaction by beta -gal assay). Similar results were obtained using other combinations of these interactors (data not shown). Finally, the specificity of this dissociator assay was demonstrated by a lack of effect of dissociator expression on two unrelated interacting pairs (Max-Mxi1 and PreS2-B31). Taken together, the interacting domains among HBX, PSMA7, and PSMC1, as characterized by the yeast two-hybrid system, were confirmed by the modified yeast two-hybrid dissociator system. In general, this modified yeast two-hybrid dissociator system could provide valuable information regarding the structural relationship among multiple interacting proteins.


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  		Fig. 3.   Characterization of specific interactions among HBX, PSMA7, and PSMC1 using a modified yeast two-hybrid dissociator system. A, this system is schematically shown here and discussed under "Results." B, four pairs of interacting proteins, one fused to the LexA DNA-binding domain (pEG202) and the other to the acidic activator B42 (pRF25) were transformed into yeast strain EGY40 containing lacZ reporter JK103 and selected on Sc-L-H-U-Glu plates. Transformants that showed expected interactions were retransformed with pRF4-6NL plasmid with or without the dissociator gene as indicated. The transformants were selected on Sc-L-H-U-T-Glu, streaked on Sc-L-H-U-T-Gal to induce the production of the diruptor, and assayed for beta -gal activities (10 colonies were assayed for each condition). The results (means ± standard deviations) are representative of at least three separate experiments. CTRL, control.

HBX Is a Coactivator of AP-1 and VP16 but Has No Effect on GCN4 and Gal4-- HBX functions as a coactivator of AP-1 and many other transcriptional factors in a variety of mammalian cells (7-9), but appears to have no effect on basal transcription (8, 24). However, it is not clear whether HBX has similar effect on transcription in yeast. Therefore, we compared the HBX transactivation activities in human hepatoma HepG2 cells and yeast. First, we studied the transactivating effect of HBX on general transcription involving a complex promoter (RSV-Luc reporter) as well as on specific transcriptional activator AP-1 in HepG2 cells and GCN4 in yeast. Previous studies suggested that yeast transactivator GCN4 is the yeast ortholog of AP-1 (28, 29). In HepG2 cells, HBX is capable of transactivating RSV-Luc and AP-1-CAT (Fig. 4A) reporter plasmids in a dose-dependent manner without exhibiting the "squelching" phenomenon. In yeast strains KNY14 (GCN4-) and KNY24 (GCN4+), expression of HBX had no effect on GCN4-responsive reporter B2079 (Fig. 4B).


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  		Fig. 4.   Effect of HBX on transcriptional activators in HepG2 cells and yeast. A, RSV and AP-1 reporter plasmids in HepG2. Increasing amounts of pCDHBX and 0.5 µg of RSV-Luc or AP-1-CAT reporter gene were transiently transfected in triplicates into HepG2 cells plated on six-well dishes. The cells were harvested 2 days later for luciferase and CAT assays. B, transactivator GCN4 in yeast. The HBX gene was cloned into expression vector pYepLac181 and cotransformed with the GCN4-responsive reporter B2079 into two yeast strains, KNY14 (GCN4-) and KNY 24 (GCN4+). Transformants were selected on Sc-L-T plates and assayed for beta -galactosidase activities (10 colonies were assayed for each condition). C, acidic activator VP16 in HepG2. HepG2 cells in six-well dishes were transfected in triplicates with increasing amounts (0.2-1.4 µg) of Gal4DB-VP16 plasmids with or without a fixed amount (0.05 µg) of HBX. 0.5 µg of reporter (p17X4TATA-CAT) was cotransfected and the total cotransfected DNA was 2 µg. CAT activities were determined 2 days after transfection. D, acidic activator Gal4 in yeast. pCL1 plasmid expressing GAL4 and p2.5HBX expressing HBX were transformed into yeast strain MaV103 and selected on Sc-L-T plate. The transformants were then assayed for beta -gal activities (10 colonies were assayed for each condition). HBX expression in the transformed yeast was demonstrated by Western immunoblotting in both experiments (data not shown). The results represent the means ± standard deviations and are representative of three separate experiments. CTRL or Ctrl, control.

Next, we studied the prototypic mammalian acidic activator VP16 and its counterpart in yeast, Gal4. In mammalian cells, expression of Gal4DB-VP16 activates reporter construct containing Gal4 DNA binding motif. We reasoned that coexpression of HBX would further increase reporter activity if HBX is indeed a coactivator of VP-16. However, HBX is also capable of activating the CMV promoter that is used to express the Gal4DB-VP16 and therefore may result in an increased level of Gal4DB-VP16, which, in turn, can lead to further increase in reporter activity. Conversely, high levels of transactivator could cause squelching with reduction of reporter activity. In order to distinguish between the two possibilities, we transfected increasing amounts of Gal4DB-VP16 plasmids with or without a fixed amount of HBX and tested for reporter activities in each condition. Transfection of the Gal4DB-VP16 plasmid with the Gal4-responsive reporter demonstrated increasing transactivation with incremental amounts of Gal4DB-VP16, but at the highest amount of transfected plasmid, classical squelching occurred with reduced reporter activity (Fig. 4C). On the other hand, cotransfection of HBX with increasing amounts of Gal4DB-VP16 led to further increase of reporter activities at all levels of Gal4DB-VP16 plasmid, including the highest level where the squelching occurred. We reasoned that if HBX functions only to transactivate the CMV promoter driving the Gal4DB-VP16 activator, one would expect squelching to occur at a lower level of transfected Gal4DB-VP16 plasmid in HBX-cotransfected cells. By contrast, HBX appeared to relieve squelching and resulted in further transactivation at high levels of transfected Gal4DB-VP16 plasmid, suggesting that HBX is indeed a coactivator of VP16. In yeast, expression of HBX had no effect on Gal4-responsive reporter activities (Fig. 4D). Taken together, these results demonstrated that HBX is a coactivator of human AP-1 and acidic activator VP16 but exerts no effect on the yeast orthologs GCN4 and Gal4.

Effect of PSMC1 Overexpression on the Transactivation Function of HBX-- We previously demonstrated that overexpression of PSMA7 appeared to activate transcription and antisense expresion of PSMA7 was able to block transactivation by HBX (24). In order to study the functional importance of HBX-PSMC1 interaction, we tested the effect of PSMC1 overexpression on luciferase expression of the RSV-Luc reporter, as well as on the transactivation function of HBX. The reporter and increasing amounts of a PSMC1 expression construct (pCDPSMC1) were cotransfected with or without a fixed amount of pCDHBX in HepG2 cells (Fig. 5). Increasing amounts of transfected pCDPSMC1 led to progressively reduced luciferase expression in RSV-Luc-transfected cells. On the other hand, HBX coexpression appeared to activate RSV-Luc expression similarly at all levels of transfected pCDPSMC1 (Fig. 5, middle panel). Therefore, the calculated transactivation by HBX appeared to be enhanced by overexpression of PSMC1 (Fig. 5, bottom panel). Similar results were obtained with the AP-1-CAT reporter (data not shown). This is in contrast with the effect of PSMA7 overexpression, which increases the expression of reporters either with or without HBX, as shown in our previous publication (24).


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  		Fig. 5.   Effects of PSMC1 overexpression on transactivation function of HBX. RSV-Luc reporter (0.25 µg) and increasing amounts (0, 0.05, 0.1, and 0.2 µg) of pCDPSMC1 were cotransfected with (B) or without (A) 0.05 µg of pCDHBX in triplicates into HepG2 cells plated on six-well dishes (the total transfected DNA was 0.5 µg). Two days after transfection, cells were lysed and luciferase activites determined. The results are shown as luciferase activities (means ± standard deviations), or as -fold induction (C) of the RSV-Luc reporter by HBX (dividing B over A). The results are representative of three separate experiments.

Inhibition of Cellular Protein Degradation by HBX-- To assess the effect of HBX on the turnover of cellular proteins known to be degraded through the ubiquitin-dependent, proteasome-mediated pathway, HBX expression construct was cotransfected with construct expressing either a tagged version of c-Jun (CMV-HA-c-Jun) or CMV-Arg-beta -gal. The HA-c-Jun has been used to study the degradation of c-Jun by the proteasome complex (32), and the Arg-beta -gal has been shown to be a proteasome substrate based on the N-end rule (33). In the pulse-chase experiment (Fig. 6A), the turnover of c-Jun was significantly inhibited from a half-life of 45 min to over 2 h. Similar observation was made with the turnover of Arg-beta -gal (Fig. 6B); its half-life was prolonged from 4.5 h to over 10 h in the presence of HBX expression.


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  		Fig. 6.   Inhibition of cellular protein degradation by HBX. HepG2 cells were transiently transfected with various expression constructs as indicated on the top of gel (a total of 15 µg of DNA/dish). 18 h later, cells were incubated with methionine-free medium for 30 min, pulse-labeled with [35S]methionine (200 µCi/ml) for 20 min, and followed by chase with medium containing excessive cold methionine (50 µg/ml). Cells were lysed at the end of the labeling and various times during chase as indicated. In B, one set of the cells transfected with Arg-beta -gal were exposed to 10 µM MG132, a specific proteasome inhibitor (41), starting 1 h prior to the pulse-labeling and continuing during the chase. The cell lysates were immunoprecipitated by appropriate antibodies (for A, the monoclonal antibody 12CA5 specific for the HA epitope; for B, anti-beta -gal antibodies). The control represents time 0 lysate of transfected cells incubated with mouse IgG. The immunoprecipitates were electrophoresed on 10% SDS-PAGE. The gel was dried and the levels of protein expression analyzed with a phosphorimager. The values of signals at time 0 were arbitrarily set at 100, and the signal intensity of other time points were adjusted accordingly. The half-lives (t1/2) are indicated on the graph.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our previous study demonstrated the specific interaction of HBX and an alpha  proteasome subunit PSMA7 (24). We further showed that HBX binds to the proteasome complex in vivo, and this interaction leads to inhibition of the chymotryptic peptidase and protease activities of the proteasome (41). In this study, we showed that HBX also interacts with another proteasome subunit PSMC1, which is a subunit of the 19 S regulatory component of the 26 S proteasome complex. These interactions were also corroborated by in vitro binding assays. Using deletion analyses in the standard yeast two-hybrid system and a modified yeast two-hybrid dissociator system, the interacting domains of HBX, PSMA7, and PSMC1 were defined. HBX was also shown to have no or little interaction with the yeast PSMA7 and PSMC1, respectively.

These interactions, as modeled in Fig. 7, predict a structural relationship of these interacting factors in vivo. The PSMA7 probably interacts with PSMC1 in the native 26 S proteasome complex. HBX interaction with either factor may disrupt this native association and leads to functional alteration of the proteasome complex. This hypothesis is consistent with the three-dimensional model of the 20 S proteasome complex, whose crystallographic structure from several species has been elucidated recently (42, 43). The PSMA7, as an alpha  subunit of the 20 S proteasome, is located on the outer ring of the proteasome complex. The C terminus of PSMA7, to which both HBX and PSMC1 bind, is exposed on the outer surface of the 20 S proteasome, presumably playing a key role in interacting with other factors such as the 19 S component or the PA28 activator (44, 45). We have also recently demonstrated the formation of complexes between HBX and various forms of the proteasome in vivo (41), further supporting this predicted structural relationship.


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  		Fig. 7.   Structural interaction between HBX and the 26-proteasome complex. The 20 S core consists of four stacked heptamer rings of alpha  or beta  subunits in the order of alpha , beta , beta , alpha  with the alpha  ring in contact with the 19 S component. HBX presumably interacts with the PSMA7 and PSMC1 subunits at the site of contact between the 20 S and the 19 S complexes.

The PSMC1 subunit belongs to a family of ATPases including several other subunits of the 19 S regulatory factor, which share 40-60% identity among one another. These ATPase subunits (Tbp-1, Tbp-7, MSS1, Sug1/Trip1) have been structurally and functionally linked to well known transcriptional activators. Tbp-1 and its closely related Tbp-7 physically interact with the Tat transactivator of human immunodeficiency virus-I, resulting in alteration of the transactivation function of Tat (46, 47). MSS1, a human gene originally identified to regulate G1 cyclins in yeast, has also been shown to be a positive modulator of Tat-mediated transactivation (48). Finally, the Sug1 gene of S. cerevisiae functions as a cofactor of Gal4 in transcriptional activation (49, 50), and its mammalian homolog Trip1 exhibits a ligand-dependent association with members of nuclear receptor family including thyroid hormone receptor, which regulates a variety of gene expression (51). The precise molecular mechanism underlying many of these functional interactions have not yet been established. It remains controversial as to whether these findings, including our observation of HBX-PSMC1 interaction and HBX-mediated transactivation, are due to a direct action of these ATPases on transcriptional activation or an indirect effect through the proteolytic function of the proteasome complex. Our recent data on HBX inhibition of proteolytic activities of proteasome suggest that the function of HBX as a coactivator of many transcriptional factors may be an indirect result of altered proteolysis (41). In this paper, the prolongation of c-Jun half-life in vivo in the presence of HBX, although it can be explained by the alternative effect of signal tranduction by HBX (52), is also consistent with this explanation. In addition, the similar effect of HBX expression on degradation of the Arg-beta -gal, which is a specific substrate of ubiquitin-proteasome pathway according to the N-end rule (33), lends further support to our hypothesis.

The PSMC1 may be somehow linked to the degradation of a general transcriptional factor; HBX, through its interaction with PSMA7 and PSMC1, possibly abrogates this effect and results in accumulation of this factor, leading to enhanced general transcription. This is consistent with our observation that overexpression of PSMC1 appears to inhibit transcriptional activity and such an effect is abrogated by coexpression of HBX. It is interesting to note that the Tat interactor Tbp-1 suppresses Tat-mediated transactivation in vivo (48). Alternatively, the interaction of HBX with these proteasome subunits could lead to another indirect, nonproteolytic pathway of transcriptional activation. The 19 S proteasome regulatory complex is closely related to the COP9 signalsome, which is a macromolecular cellular complex involved in signal transduction (53-55). This complex has been shown to phosphorylate Ikappa B-alpha , p105 precursor of NFkappa B, and c-Jun and many of its subunits contained canonical MAP kinase kinase sites (56). This notion is also supported by the reported effects of HBX on signal transduction (57).

Although HBX has been proposed to interact directly with various transcriptional factors in vitro (12, 13, 16, 40, 58, 59), the existence of a functional complex between HBX and cellular transcriptional machinery in vivo has not been shown. Studies on cellular localization of HBX showed HBX to be predominantly a cytoplasmic protein (10, 60). In addition, HBX with a genetically engineered nuclear localization signal was actually defective for much of its transactivation function (10). Studies on signal transduction by HBX (57) as well as our data on HBX also supported that HBX probably acts through an indirect mechanism on transcription. The observation that HBX does not exhibit squelching by itself in HepG2 (41) and can further coactivate transcriptional activators under squelching condition suggests that HBX probably does not interact directly with either basal or activated transcriptional machinery in a way that many transcription activators do. HBX probably functions as an indirect coactivator that somehow alters the functional capacity of cellular transcription. This may explain why HBX is capable of promiscuously activating a multitude of promoters. However, it is possible that HBX, in small quantity, may translocate to the nucleus to interact with a specific subset of transcriptional factors, such as CREB/ATF, RPB5, and TFIIH, and simultaneously mediates other pleiotropic effects through an indirect mechanism(s) in the cytoplasm. The two mechanisms are not necessarily mutually exclusive.

Finally, HBX does not appear to function as a coactivator of transcription in yeast under our experimental condition. Although there are many possibilities that may explain this observation, it is tempting to think that the reason could lie in the inability of HBX to interact with the yeast proteasome subunits. It would be interesting to replace the yeast PSMA7 and/or PSMC1 subunits with those of human and to test whether one might restore the function of HBX in yeast. If this was possible, it would be a direct proof that the proteasome mediates the transactivation function of HBX.

    ACKNOWLEDGEMENTS

We thank Sophia Tsai, Robert Kingston, Mathhias Treier, Alexander Varshavsky, and David M. Rubin for providing plasmids and Alan Hinnebusch for technical advice and providing plasmids and yeast strains.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Liver Diseases Section, NIDDK, National Institutes of Health, 10 Center Dr., Rm. 9B16, Bethesda, MD 20892-1800. Tel.: 301-496-1721; Fax: 301-402-0491; E-mail: jliang@nih.gov.

Published, JBC Papers in Press, March 15, 2000, DOI 10.1074/jbc.M910378199

2 A. Furusaka and T. J. Liang, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HBV, hepatitis B virus; HBX, hepatitis B virus X protein; beta -gal, beta -galactosidase; aa, amino acid(s); CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HA, hemagglutinin; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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