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

• DOI: 10.1074/jbc.m910378199
• Publication Date: 2002-07-26T14:55:13Z
• Authors: Zhensheng Zhang; Nobuyuki Torii; Akihiro Furusaka; Navara Malayaman; Zongyi Hu; T.Jake Liang
• Keywords: Characterization

Hepatitis B virus (HBV) has a unique fourth open reading frame coding for 16.5-kDa protein known as hepatitis X (HBX). The importance of HBX in the life cycle HBV been well established, but underlying molecular function remains controversial. We previously identified proteasome subunit PSMA7 that interacts specifically with Saccharomyces cerevisiae two-hybrid system. Here we demonstrate PSMC1, an ATPase-like 19 S component, also and PSMA7. Analysis interacting domains among PSMA7, by deletion site-directed mutagenesis suggested mutually competitive structural relationship these polypeptides. nature interactions is further demonstrated using modified yeast dissociator crucial sequences involved interaction PSMC1 are important its transcriptional coactivator. HBX, while functioning coactivator AP-1 acidic activator VP-16 mammalian cells, had no effect on transactivation their functional orthologs GCN4 Gal4 yeast. Overexpression seemed to suppress expression various reporters cells; this effect, however, was overcome coexpression HBX. In addition, inhibited cellular turnover c-Jun ubiquitin-Arg-β-galactosidase, two substrates ubiquitin-proteasome pathway. Thus, complex metazoan cells may underlie basis target β-galactosidase amino acid(s) chloramphenicol acetyltransferase polyacrylamide gel electrophoresis polymerase chain reaction hemagglutinin cytomegalovirus Human (HBV)1 belongs group hepadnaviruses includes viruses woodchuck, ground squirrel, tree Pekin duck, heron. frame, termed (HBX) gene. gene conserved codes (1.Haruna Y. Hayashi N. Katayama K. Yuki 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. 14: 29-37Crossref (100) Scholar). can activate transcription variety viral genes (3.Aufiero B. Schneider R.J. EMBO J. 1990; 9: 497-504Crossref (129) 4.Colgrove R. Simon G. Ganem D. 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Liang T.J. 1996; 70: 5582-5591Crossref present study, another HBX-interacting clone which member regulatory factor (25.Dubiel W. Ferrell Pratt Reichsteiner 267: 22699-22702Abstract 26.Dubiel Rechsteiner Rep. 21: 27-34Crossref (123) 27.Coux O. Tanaka Goldberg A.L. Annu. Rev. Biochem. 65: 801-847Crossref (2215) were characterized, specificity evaluated studied yeasts, HBX-PSMC1 interaction. Four strains used, genetic backgrounds summarized TableI. EGY48 used standard system, EGY40 KNY14 (inactivated gene) KNY24 (wild-type GCN4) (28.Hinnebusch A.G. Lucchini Fink G.R. 1985; 82: 498-502Crossref (70) 29.Hinnebusch 5: 2349-2360Crossref (152) study GCN4, ortholog. MaV103 strain test transactivator.Table IGenetic background strainsYeast strainGenetic backgroundEGY48Mata, leu2, his3, trp1, ura3, LexAop-LEU2EGY40Mata, ura3KNY14Mata, gcn4–103KNY14Mata, Open table new tab For fused lexA binding domain pEG202 pEG202HBX reporter construct lexAop-lacZgene, permits determination based (β-gal) activity. Two lacZ constructs, pSH18–34 JK103 (both URA3 marker), contained eight LexA sites, respectively (23.Gyuris pRF4–6NL, TRP1 plasmid containing Gal1 promoter galactose-inducible, “dissociator” (30.Colas Cohen Jessen Grishina McCoy 380: 548-550Crossref (373) pCL1, LEU2 expressing GAL4, p2.5 theHIS3 marker described 31.$$$$$$ ref data missingGoogle p2.5HBX generated inserting fragment (EcoRI-NotI) from into plasmid. Mutations introduced PCR-based (QuikChange kit, Stratagene, La Jolla, CA). mutants HBXmd HBXsm, middle small respectively, PCR. Deletion convenient restriction sites or PCR (for map, see Fig. 2). Chimeric constructs human (YHA7.169, YHA7.199, YHA7.227) PCR, exchanging corresponding regions sequences. All mutant confirmed sequencing. pYepHBX ligation ADH1 (PstI-HindIII pJG7–1) (31.$$$$$$ aHindIII-BamHI JG4–6HBX terminator PstI andBamHI pYepLac181 (provided Alan Hinnebusch, NICHD, National Institutes Health, Bethesda, MD). Plasmid B2079 (TRP1 marker) GCN4-responsive reporter, provided Hinnebusch. p17X4TATA-CAT, contains four copies thymidine kinase minimal (CAT) reporter; pCEP4GLVP fusion activation driven SV40 promoter, Sophia Tsai (Baylor College Medicine, Houston, TX). Robert Kingston (Massachusetts General Hospital, Boston, MA), respectively. CMV-HA-c-Jun full-length tagged influenza (HA) epitope at N terminus Mathias Treier (European Molecular Biology Laboratory, Heidelberg, Germany) (32.Treier Staszewski L.M. Bohmann 78: 787-798Abstract (845) CMV-Arg-β-gal ubiquitin-Arg-β-galactosidase Alexander Varshavsky, California Institute Technology, Pasadena, CA) (33.Varshavsky 725-735Abstract (388) pCDNA1. vitro experiment, [35S]Met-labeled proteins Rabbit Reticulocyte Extract (Promega, Madison, WI). (full-length) (aa 137–248) cloned pGEX-KG vector (Amersham Pharmacia Biotech) expressed glutathione S-transferase (GST) protein, purified glutathione-coupled agarose beads. translated then incubated protein-bound beads NETN buffer (20 mm Tris, pH 8.0, EDTA 1 mm, 100 mmNaCl, 0.5% Nonidet P-40) room temperature h constant mixing. washed extensively same bound subjected 15% SDS-PAGE PhosphorImager (Storm, Dynamics, Sunnyvale, analysis. pulse-chase lysed directly 10-cm dish ml cold lysis 50 mmTris-HCl, 7.4, 150 NaCl, Triton X-100, μg/ml leupeptin, aprotinin, PMSF. centrifuged 13,000 × g 15 min 4 °C remove nuclei other insoluble cell debris. Immunoprecipitation performed incubating lysates antibody first G-coupled Sepharose (Santa Cruz Biotechnology, Inc., Santa Cruz, immunoprecipitates separated analyzed PhosphorImager. monoclonal 12CA5 specific HA anti-β-gal purchased Roche Biochemicals. Standard pairs proteins, HBX-PSMA7, PSMC1.1-PSMA7, Max-Mxi1 (34.Zervos A.S. Gyuris 72: 223-232Abstract (661) PreS2-B31 2A. Furusaka Liang, unpublished data. (pEG202: promoter,HIS3 B42 (pRF25: Yeast transformed thelacZ JK103. Positive clones retransformed pRF4–6NL (Gal1 promoter,LEU2 marker). transformants grown plates Glu/CSM-His-Leu-Trp-Ura (Bio 101, Vista, 30 2–3 days until colonies visible. 10 independent each streaked onto Gal/CSM-His-Leu-Trp-Ura (galactose dissociator) days. harvested, acid-washed 425–600-μm glass (Sigma), assayed β-gal activities Galacto-Light kit (Tropix, Bedford, MA). Three plasmids assays HepG2 cells. RSV-Luc, AP-1-CAT (containing adjacent metallothionein IIA promoter), mTK-Luc promoter) hepatoma Dulbecco's Eagle's medium (Life Technologies, Inc.) 10% fetal bovine serum humidified incubator (5% CO2). Transient transfection 35-mm carried out (5 Prime → 3 Prime, Boulder, CO). Luciferase assay Monolight luminometer (Analytical Luminescence San Diego, CAT enzyme-linked immunosorbent (Roche Biochemicals). several interacted One strongly complex, clones, component 26 To subunitsin vitro, constructed GST plasmids, one PSMC1. bacteria translated, proteins. polypeptides analysis (Fig. 1). GST-PSMA7 -PSMC1 GST. Similar between GST-PSMC1 -HBX. appeared stronger than This finding consistent relative strength results corroborated characterize cDNA tested our previous determined C-terminal region additional (Fig.2 A). H7.198 137–198), H7.227 137–227), H7.230 137–230) exhibited either H7.237 137–237) retained nearly full N-terminally truncated H7.169 169–248) similar 2 ortholog (YPSM7) (35.Remacha Saenz-Robles M.T. Vilella M.D. Ballesta 263: 9094-9101Abstract YPSMA7 weak A), suggesting exhibits selective three chimeric PSMA7: YH7.169, YH7.199, YH7.227. YH7.169 YH7.199 YH7.227 On hand, HY7.169 did, weaker YH7.169. Taken together, aa 199–237 necessary information both However, observed minor difference mapping slightly different. Comparison shows major divergence probably accounts binding. showed 1–440) PSMC1.1 123–440) although much higher deletions, PSMC1.2 123–316) PSMC1.3 317–440), contrast, high PSMC1.2. Collectively, distinct domains: N-terminal (36.Heinemeyer Trondle Albrecht Wolf D.H. Biochemistry. 33: 12229-12237Crossref (102) weakly moderate pairs, (data shown). Sequence subunits again account Functional defined 24.Huang 37.Runkel Fischer Schaller Virology. 197: 529-536Crossref (54) These appear overlap protease inhibitor WHVX Our established association mutations second respect PSMA7; defective negative determine experiments C). revealed H139D abolished C137S R138Q reduced markedly Together current suggests albeit motifs exactly same. gene, alternative translation initiations, could potentially encode differentially transactivate II III promoters (38.Kwee Aufiero 66: 4382-4389Crossref From above, reason all forms should equally third in-frame start codons, generated. Interactions HBXsm despite scoring RSV-Luc subunits. domain, subunits, factor(s) Recent reports repair enzyme UVDDB (39.Sitterlin Lee Prigent Tiollais Transy 71: 6194-6199Crossref general TFIIH 40.Haviv Vaizel 3413-3420Crossref (87) define adopted diagrammatically Fig.3 Briefly, partners (bait prey) under (pRF4–6) (lacZ reporter) after induction galactose. If bait prey domains, inhibition original would result, leading decreased groups interactors (Fig.3 B). HBX-PSMA7 unrelated PreS2-B31, controls studied. instead because it conferred expected competed PSMC1.1. As predicted, (to 71% activity) more significantly 55%) Similarly, PSMC1.1-PSMA7 dramatically 47% only modestly 75%). relatively compared (about 30% assay). obtained combinations Finally, lack (Max-Mxi1 PreS2-B31). characterized general, provide valuable regarding multiple functions many factors