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• W2034975078 abstract "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-β-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. 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-β-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. hepatitis B virus hepatitis B virus X protein β-galactosidase amino acid(s) chloramphenicol acetyltransferase polyacrylamide gel electrophoresis polymerase chain reaction hemagglutinin cytomegalovirus 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.Haruna Y. Hayashi N. Katayama K. Yuki N. Kasahara A. Fusamoto H. Kamada T. Hepatology. 1991; 13: 417-421Crossref PubMed Scopus (24) Google Scholar, 2.Wang W.L. London W.T. Lega L. Feitelson M.A. Hepatology. 1991; 14: 29-37Crossref PubMed Scopus (100) Google Scholar). The protein can activate the transcription of a variety of viral and cellular genes (3.Aufiero B. Schneider R.J. EMBO J. 1990; 9: 497-504Crossref PubMed Scopus (129) Google Scholar, 4.Colgrove R. Simon G. Ganem D. J. Virol. 1989; 63: 4019-4026Crossref PubMed Google Scholar, 5.Zahm P. Hofschneider P.H. Koshy R. Oncogene. 1988; 3: 169-177PubMed Google Scholar) and induce liver cancer in certain transgenic mouse model (6.Kim C.M. Koike K. Saito I. Miyamura T. Jay G. Nature. 1991; 351: 317-320Crossref PubMed Scopus (1043) Google Scholar). 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.Haviv I. Vazl D. Shaul Y. Mol. Cell. Biol. 1995; 15: 1079-1085Crossref PubMed Google Scholar, 8.Natoli G. Avantaggiati M.L. Chirillo P. Costanzo A. Artini M. Balsano C. Levrero M. Mol. Cell. Biol. 1994; 14: 989-998Crossref PubMed Scopus (125) Google Scholar, 9.Seto E. Mitchell P.J. Ten T.S. Nature. 1990; 344: 72-74Crossref PubMed Scopus (212) Google Scholar) and to activate various signal transduction pathways (10.Doria M. Klein N. Lucito R. Schneider R.J. EMBO J. 1995; 14: 4747-4757Crossref PubMed Scopus (274) Google Scholar, 11.Wang H.D. Trivedi A. Johnson D.L. Mol. Cell. Biol. 1998; 18: 7086-7094Crossref PubMed Scopus (76) Google Scholar). Several recent studies have also identified possible cellular targets of HBX, including members of the CREB/ATF family (12.Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (377) Google Scholar, 13.Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (146) Google Scholar), the TATA-binding protein (14.Qadri I. Maguire H.F. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1003-1007Crossref PubMed Scopus (185) Google Scholar), RNA polymerase subunit RPB5 (15.Lin Y. Nomura T. Cheong J. Dorjsuren D. Iida K. Murakami S. J. Biol. Chem. 1997; 272: 7132-7139Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16.Cheong J. Yi M. Lin Y. Murakami S. EMBO J. 1995; 14: 143-150Crossref PubMed Scopus (239) Google Scholar), the UV-damaged DNA-binding protein (17.Lee T.H. Elledge S.J. Butel J. J. Virol. 1995; 69: 1107-1114Crossref PubMed Google Scholar), and the replicative senescence p55 sen (18.Sun B.S. Zhu X. Clayton M.M. Pan J. Feitelson M.A. Hepatology. 1998; 27: 228-239Crossref PubMed Scopus (44) Google Scholar). HBX has also been shown to interact with p53 and inhibit its function (19.Truant R. Antunovic J. Greenblatt J. Prives C. Cromlish J.A. J. Virol. 1995; 69: 1851-1859Crossref PubMed Google Scholar, 20.Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (629) Google Scholar). 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.Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar). Using the Saccharomyces cerevisiae two-hybrid system (22.Fields S. Song O.-K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4799) Google Scholar,23.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1316) Google Scholar), we previously identified an α 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.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). 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.Dubiel W. Ferrell K. Pratt G. Reichsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar, 26.Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 21: 27-34Crossref PubMed Scopus (123) Google Scholar, 27.Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2215) Google Scholar). 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. Four S. cerevisiae strains were used, and their genetic backgrounds are summarized in TableI. 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.Hinnebusch A.G. Lucchini G. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 498-502Crossref PubMed Scopus (70) Google Scholar, 29.Hinnebusch A.G. Mol. Cell. Biol. 1985; 5: 2349-2360Crossref PubMed Scopus (152) Google Scholar) 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.Table IGenetic background of S. cerevisiae strainsYeast strainGenetic backgroundEGY48Mata, leu2, his3, trp1, ura3, LexAop-LEU2EGY40Mata, leu2, his3, trp1, ura3KNY14Mata, leu2, trp1, ura3, gcn4–103KNY14Mata, leu2, trp1, ura3, GCN4 Open table in a new tab For the yeast two-hybrid system, the HBX gene was fused to the lexA DNA binding domain of pEG202 as pEG202HBX (24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). The reporter construct was lexAop-lacZgene, which permits determination of interaction based on β-galactosidase (β-gal) activity. Two lacZ reporter constructs, pSH18–34 and JK103 (both with URA3 marker), contained eight and two LexA binding sites, respectively (23.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1316) Google Scholar). 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.Colas P. Cohen B. Jessen T. Grishina I. McCoy J. Brent R. Nature. 1996; 380: 548-550Crossref PubMed Scopus (373) Google Scholar). pCL1, a LEU2 plasmid expressing GAL4, and p2.5 plasmid containing theHIS3 marker have been described (22.Fields S. Song O.-K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4799) Google Scholar, 31.$$$$$$ ref data missingGoogle Scholar). p2.5HBX was generated by inserting HBX fragment (EcoRI-NotI) from pEG202HBX into p2.5 plasmid. Mutations in HBX were introduced by PCR-based mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA). The HBX mutants have been described previously (24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar) and HBXmd and HBXsm, containing the middle and small HBX, respectively, were generated by PCR. Deletion mutants of PSMA7 and PSMC1 were generated using convenient restriction sites or by PCR (for map, see Fig. 2). Chimeric constructs of yeast and human PSMA7 constructs (YHA7.169, YHA7.199, and YHA7.227) were generated by PCR, exchanging corresponding regions of yeast with human sequences. All mutant constructs were confirmed by DNA sequencing. pYepHBX was generated by ligation of the ADH1 promoter (PstI-HindIII fragment from pJG7–1) (31.$$$$$$ ref data missingGoogle Scholar) and aHindIII-BamHI fragment from JG4–6HBX containing HBX and ADH1 transcription terminator into the PstI andBamHI sites of a LEU2 plasmid pYepLac181 (provided by Alan Hinnebusch, NICHD, National Institutes of Health, Bethesda, MD). Plasmid B2079 (TRP1 marker) (28.Hinnebusch A.G. Lucchini G. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 498-502Crossref PubMed Scopus (70) Google Scholar), 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.Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (845) Google Scholar). The CMV-Arg-β-gal plasmid was generated by inserting the DNA fragment coding for ubiquitin-Arg-β-galactosidase (provided by Alexander Varshavsky, California Institute of Technology, Pasadena, CA) (33.Varshavsky A. Cell. 1992; 69: 725-735Abstract Full Text PDF PubMed Scopus (388) Google Scholar) into the plasmid pCDNA1. 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 mmNaCl, 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 mmTris-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-β-gal monoclonal antibody were purchased from Roche Molecular Biochemicals. Standard yeast two-hybrid system was performed as described (23.Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1316) Google Scholar). For the modified yeast two-hybrid dissociator system, four pairs of interacting proteins, HBX-PSMA7, PSMC1.1-PSMA7, Max-Mxi1 (34.Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (661) Google Scholar), and PreS2-B31 2A. Furusaka and T. J. Liang, unpublished data. 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.Colas P. Cohen B. Jessen T. Grishina I. McCoy J. Brent R. Nature. 1996; 380: 548-550Crossref PubMed Scopus (373) Google Scholar), respectively. Yeast strain EGY40 was first transformed with these interacting pairs and then thelacZ 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 β-gal activities using the Galacto-Light kit (Tropix, Bedford, MA). 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.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). 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 → 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). Using the yeast two-hybrid system, we previously identified several independent clones that interacted specifically with HBX (24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). One strongly interacting clone was identified as an α subunit of the proteasome complex, PSMA7 (24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). One of the other clones, PSMC1, was an ATPase-like subunit of the 19 S regulatory component of the 26 S proteasome complex (25.Dubiel W. Ferrell K. Pratt G. Reichsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar, 26.Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 21: 27-34Crossref PubMed Scopus (123) Google Scholar). 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 subunitsin 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. 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.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). 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.2 A). 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.2 A). In addition, the N-terminally truncated construct H7.169 (aa 169–248) had binding activity similar to that of H7.237 (Fig. 2 A). Since the yeast PSMA7 ortholog (YPSM7) (35.Remacha M. Saenz-Robles M.T. Vilella M.D. Ballesta J.P. J. Biol. Chem. 1988; 263: 9094-9101Abstract Full Text PDF PubMed Google Scholar) has regions of sequence homology with the PSMA7, its interaction with HBX and PSMC1 was analyzed (Fig. 2 A). The YPSMA7 demonstrated no binding to HBX but had a weak binding to PSMC1 (Fig. 2 A), 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. 2 A). 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. Fig. 2 B 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.Heinemeyer W. Trondle N. Albrecht G. Wolf D.H. Biochemistry. 1994; 33: 12229-12237Crossref PubMed Scopus (102) Google Scholar) 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. Functional mapping of HBX has defined two structural domains that are crucial for the transactivation function of HBX (21.Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar, 24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar, 37.Runkel L. Fischer M. Schaller H. Virology. 1993; 197: 529-536Crossref PubMed Scopus (54) Google Scholar). These two domains appear to overlap with the putative Kunitz-type domain of protease inhibitor that is present in both HBX and WHVX (21.Arii M. Takada S. Koike K. Oncogene. 1992; 7: 397-403PubMed Google Scholar, 24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). 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.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar). To determine the interacting domain of HBX with PSMC1, we carried out similar experiments using various HBX mutants (Fig. 2 C). 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. 2 C). Together with our previous data on PSMA7 and HBX interaction (24.Huang J. Kwong J. Sun E.C. Liang T.J. J. Virol. 1996; 70: 5582-5591Crossref PubMed Google Scholar), 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.Kwee L. Lucito R. Aufiero B. Schneider R.J. J. Virol. 1992; 66: 4382-4389Crossref PubMed Google Scholar). 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. 2 C). 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.Sitterlin D. Lee T.H. Prigent S. Tiollais P. Butel J.S. Transy C. J. Virol. 1997; 71: 6194-6199Crossref PubMed Google Scholar), the RNA polymerase subunit RPB5 (15.Lin Y. Nomura T. Cheong J. Dorjsuren D. Iida K. Murakami S. J. Biol. Chem. 1997; 272: 7132-7139Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 16.Cheong J. Yi M. Lin Y. Murakami S. EMBO J. 1995; 14: 143-150Crossref PubMed Scopus (239) Google Scholar), and the general transcriptional factor TFIIH (14.Qadri I. Maguire H.F. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1003-1007Crossref PubMed Scopus (185) Google Scholar, 40.Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar). 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.Colas P. Cohen B. Jessen T. Grishina I. McCoy J. Brent R. Nature. 1996; 380: 548-550Crossref PubMed Scopus (373) Google Scholar). The system is diagrammatically summarized in Fig.3 A. 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 β-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 β-gal activity. In the present study, four groups of interactors were evaluated using this system (Fig.3 B). Two interacting pairs from this study, HBX-PSMA7 and PSMC1.1-PSMA7, and two unrelated interacting pairs, Max-Mxi1 (34.Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (661) Google Scholar) 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. 3 B). 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 β-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. HBX functions as a coactivator of AP-1 and many other transcriptional factors in a variety of mammalian cells (7.Haviv I. Vazl D. Shaul Y. Mol. Cell. Biol. 1995; 15: 1079-1085Crossref PubMed Google Scholar, 8.Natoli G. Avantaggiati M.L. Chirillo P. Costanzo A. Artini M. Balsano C. Levrero M. Mol. Cell. Biol. 1994; 14: 989-998Crossref PubMed Scopus (125) Google Scholar, 9.Seto E. Mitchell P.J. Ten T.S. Nature. 1990;" @default.
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