Proteasome Activator PA28{gamma}-Dependent Nuclear Retention and Degradation of Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) core protein plays an important rolein the formation of the viral nucleocapsid and a regulatoryprotein involved in hepatocarcinogenesis. In this study, wehave identified proteasome activator PA28 ${gamma}$ (11S regulator ${gamma}$ ) asan HCV core binding protein by using yeast two-hybrid system.This interaction was demonstrated not only in cell culture butalso in the livers of HCV core transgenic mice. These findingsare extended to human HCV infection by the observation of thisinteraction in liver specimens from a patient with chronic HCVinfection. Neither the interaction of HCV core protein withother PA28 subtypes nor that of PA28 ${gamma}$ with other Flavivirus coreproteins was detected. Deletion of the PA28 ${gamma}$ -binding region fromthe HCV core protein or knockout of the PA28 ${gamma}$ gene led to theexport of the HCV core protein from the nucleus to the cytoplasm.Overexpression of PA28 ${gamma}$ enhanced the proteolysis of the HCV coreprotein. Thus, the nuclear retention and stability of the HCVcore protein is regulated via a PA28 ${gamma}$ -dependent pathway throughwhich HCV pathogenesis may be exerted.

INTRODUCTION

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Introduction

Hepatitis C virus (HCV) is the causative agent in most casesof acute and chronic non-A, non-B hepatitis (16, 51). Over 50%of patients with acute infection evolve into a chronic carrierstate (26), and persistent infection frequently results in chronichepatitis. Chronic HCV infection may lead to the developmentof cirrhosis and eventually hepatocellular carcinoma (21, 51).HCV belongs to the Flaviviridae family, a family that also includesJapanese encephalitis virus (JEV) and Dengue fever virus (DEN),and possesses a viral genome consisting of a single positive-strandRNA of approximately 9.6 kb and encoding approximately 3,000amino acids in a single polypeptide (9, 58). HCV proteins areproduced as a single polypeptide that is posttranslationallycleaved by host cellular peptidases and viral proteases to yieldat least 10 viral proteins (7, 10, 12, 54).

A comparison of the genome structure of HCV with other flaviviruses,as well as the observation of a specific interaction of viralsense RNA with HCV core protein in cells (53, 68), suggeststhat the HCV core protein forms the nucleocapsid with viralgenome RNA. An HCV core protein consisting of the N-terminal191 amino acids is generated by protein cleavage by host signalpeptidase(s) (37, 52). The HCV core protein is further processedinto a mature core protein lacking its C-terminal hydrophobicregion by either an unknown host protease (52, 65) or by a signalpeptide peptidase (36). The matured core protein is retainedon the endoplasmic reticulum (ER) either by an interaction withimmature core protein on the ER membrane (29) or via E1 envelopeprotein (32). The C-terminal hydrophobic region between aminoacids 174 and 191 is essential for HCV core protein anchoringon the ER membrane and for the signal sequence of E1 proteinto translocate into the ER lumen. Core proteins truncated atthe C termini are mainly localized in the nucleus and, to lesserextent, in the cytoplasm (8, 55). Further processing of theHCV core protein yields a 16-kDa product whose C terminus isnear amino acid 151; this protein translocates into the nucleus(30, 31, 55).

We have reported that hepatic steatosis and hepatocellular carcinomaare induced in transgenic mice expressing the HCV core protein,suggesting that the HCV core protein has an oncogenic activityin liver. These data further suggest that the cellular componentsresponsible for HCV-induced carcinogenesis exist not only inhumans but also in mice (39). Thus, the identification of core-bindingpartners in mammalian cells could potentially clarify the molecularmechanism(s) of HCV-induced hepatocarcinogenesis. Several cytoplasmicand nuclear proteins have been reported to bind the HCV coreprotein to both induce carcinogenesis and facilitate virionformation. A report has suggested that the HCV core proteinmay sequester LZIP, a putative tumor suppressor, in the cytoplasm,with a resulting enhancement of carcinogenesis of NIH 3T3 cells(18). The HCV core protein interacts with the C-terminal regionof p53 and enhances its transcriptional activity through augmentationof p53 DNA binding affinity (46). A putative cellular RNA helicase,primarily localized in the nucleus and to a lesser extent inthe cytoplasm, interacts with the N-terminal 40 amino acidsof the HCV core protein and is colocalized with the HCV coreprotein in both cellular locations (33, 67). It was recentlyreported that the HCV core protein directly binds and activatesSTAT3 by phosphorylation through a JAK-independent pathway;cells overexpressing both HCV core protein and STAT3 exhibitedanchorage-independent growth and tumorigenesis (66). These reportssuggest that the HCV core protein functions in both the nucleusand cytoplasm.

In this report, we identify proteasome activator PA28 ${gamma}$ (11S regulator)as an HCV core binding protein by the yeast two-hybrid system.It is well known that PA28 ${gamma}$ enhances the latent proteasome activityof the 20S proteasome and is predominantly localized in thenucleus (48, 62). PA28 ${gamma}$ is conserved across the animal kingdomfrom invertebrates to vertebrates (34, 47), although the biologicalsignificance of PA28 ${gamma}$ is largely unknown. Here, we demonstratethrough several lines of evidence that PA28 ${gamma}$ specifically interactswith the HCV core protein and remains in the nucleus, consequentlyregulating its stability.

MATERIALS AND METHODS

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Materials and Methods

Plasmids. Human PA28 ${gamma}$ cDNA was isolated from a human fetal brain libraryby the advanced yeast two-hybrid technique (MATCHMAKER two-hybridsystem 3; Clontech, Palo Alto, Calif.) with amino acids 1 to173 of the HCV core protein as bait. The gene encoding HCV coreprotein was amplified from HCV strain J1 (genotype 1b) (2) andcloned into the pGBKT7 vector (pGBKT7HCVCore173). The cDNA ofPA28 ${gamma}$ was amplified by PCR with Pfu turbo DNA polymerase (Stratagene,La Jolla, Calif.) and cloned into pEFFlagpGPKpuro (17), pEGFP-C3,and pDsRed2N1; the sequence was verified by DNA sequencing.The gene encoding PA28 ${gamma}$ , with amino acids 82 to 90 deleted, wasamplified by splicing the overlapping extension (13, 15) andcloned into pDsRed2N1 (Clontech). Other mutant constructs ofthe HCV core protein were introduced into pCAG-GS (45) and pEGFP-C3(Fig. 1). The genes encoding the core proteins of DEN (aminoacids 1 to 100) and JEV (amino acids 1 to 105), both lackingthe C-terminal hydrophobic regions, were amplified by PCR andcloned into pEGFP-C3. F protein was shown to be synthesizedby ribosomal frameshift of the core protein-coding sequence(64). The gene encoding F protein of the -2/+1 frame attachedto a Flag tag at N terminus was generated by deletion of oneadenine in codon 10 and then introduced into the pEFFlagpGKpurovector. The gene encoding human Bad or human FKBP attached toa hemagglutinin (HA) tag were isolated from human fetal librariesand introduced into pIRESbleo (Clontech). Mouse anti-Flag (M2),mouse anti-HA (HA.11), and mouse anti-cytochrome c oxygenasesubunit IV (20E) antibodies were purchased from Sigma (St. Louis,Mo.), Babco (Richmond, Calif.), and Molecular Probes (Eugene,Oreg.), respectively. Horseradish peroxidase-conjugated goatanti-mouse and anti-rabbit antisera were purchased from ICNPharmaceuticals (Aurora, Ohio). Rabbit antisera against syntheticpeptides corresponding to amino acids 70 to 85 of PA28 ${gamma}$ , 5 to19 of PA28 ${alpha}$ , or 15 to 31 of PA28ß were purchased fromAFFINITI (Exeter, United Kingdom).

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FIG. 1. Expression constructs of HCV core protein used in this study. The genes encoding HA-tagged HCV mutants or Flag-tagged HCV mutants were cloned into pCAG-GS vectors (45) while the genes encoding EGFP-fused proteins were introduced into pEGFP-C3. Other vectors are described in the text or in figure legends.

Yeast two-hybrid assay and library screening. The bait vector, pGBKT7HCVCore173, described above, was introducedinto Saccharomyces cerevisiae strain AH109. The yeast containingpGBKT7HCVCore173 were grown in yeast extract-peptone-dextrosemedium and transfected with library plasmids based on pACT2(Clontech), a vector encoding ampicillin resistance. Clones(2 x 10⁶) from a human fetal brain library were screened (Clontech).The yeast clones encoding pGBKT7-53 and pGADT7-T (Clontech)were used as a positive control while yeast containing pGBKT7and pGADT7 were considered the negative control. Yeast coloniesgrown on dropout plates lacking tryptophan, leucine, histidine,and adenine were inoculated on two new dropout plates lackingleucine and tryptophan. One of the two plates was subjectedto ß-galactosidase assay according to the method ofDuttweiller (11), and the other plate was kept at 4°C asa master plate. One of 85 dropout-plate-positive clones ledto dark blue staining on a ß-galactosidase assay plateto the same extent as the positive control and was called C1-24.The total DNA was recovered from C1-24 and introduced into Escherichiacoli strain JM109. The plasmid from C1-24 was recovered fromthe clones independently grown on the Luria-Bertani plate containing10 µg of ampicillin/ml. The sequences of the insert weredetermined by using the Big Dye terminator reaction mixtureand an ABI Prism 310 genetic analyzer (Applied Biosystems Japan,Tokyo, Japan). All 3 clones contained the same insert encodinghuman PA28 ${gamma}$ .

Transfection, immunoprecipitation, and immunoblotting. Plasmid vectors were transfected into HeLa or 293T cells byliposome-mediated transfection. Immunoprecipitation and liposome-mediatedtransfection were reported previously (38). Human and mouseliver samples were washed twice with cold phosphate-bufferedsaline (PBS) and then homogenized in 10 volumes of 20 mM Tris-HCl(pH 7.4) containing 135 mM NaCl, 1% Triton X-100, 10% glycerol,supplemented with 0.5 µg of Pefabloc (Pentapharm, Munich,Germany)/ml, 1 mM phenylmethylsulfonyl fluoride, 1 µgof soybean trypsin inhibitor/ml, 50 mM NaF, and 5 mM Na₃VO₄(Tris lysis buffer). Mouse monoclonal antibodies to the HCVcore protein (clones 11-4, 11-10, and 11-14) (5) were used forimmunoprecipitation and immunoblotting (38).

Laser scanning confocal microscopy. Transfectants were grown on glass slides at 37°C overnight,washed twice with PBS, and fixed with 4% paraformaldehyde for15 min at room temperature. Enhanced green fluorescent protein(EGFP) fusion protein-expressing cells were examined directly.For samples requiring immunostaining, cells were washed twicewith PBS after fixation, permeabilized for 15 min at room temperaturewith PBS containing 0.5% Triton X-100, and incubated in PBScontaining 1% bovine serum albumin (PBS-BSA) in order to blocknonspecific binding. Cells were then incubated at room temperaturefor 30 min in PBS-BSA containing 1 µg of rabbit anti-PA28 ${gamma}$ antiserum (AFFINITI)/ml and mouse anti-HA antibody. Cells werewashed three times with PBS-BSA and incubated at room temperaturefor 30 min in PBS-BSA containing 0.5 µg of Alexa Fluor488-conjugated anti-rabbit immunoglobulin G (IgG) and AlexaFluor 594-conjugated anti-mouse IgG antisera (Molecular Probes)/ml.After being washed three times with PBS-BSA, all samples werevisualized with a Bio-Rad (Tokyo, Japan) confocal laser-scanningmicroscope.

Mouse embryonic fibroblasts isolated from PA28 ${gamma}$ knockout mice. Embryonic fibroblasts from PA28 ${gamma}$ knockout mice were preparedas previously described (42). Cells were cultured at 37°C(5% CO₂) in Dulbecco's modified Eagle’s medium supplementedwith 10% fetal bovine serum, penicillin, streptomycin, sodiumpyruvate, and nonessential amino acids. Plasmid vectors weretransfected into cells after three passages, and the intracellularlocalization of proteins was examined.

Time-lapse microscopy. HeLa cells or mouse embryonic fibroblasts were seeded on 35-mm-diameterculture dishes, grown overnight to 70% confluence, and transfectedwith 2.5 µg of plasmid DNA with Lipofectamine 2000 perthe manufacturer's instructions (Invitrogen Corp., Carlsbad,Calif.). For immunofluorescent microscopy, cells were incubatedat 37°C overnight after transfection, trypsinized, reseeded,and cultivated for 20 h. For time-lapse microscopy, cells wereseeded on 35-mm-diameter dishes, grown for 24 h after transfection,and incubated in Dulbecco's modified Eagle’s medium containing10% fetal calf serum at 37°C. Cells were viewed at 37°C(5% CO₂) with an Olympus (Tokyo, Japan) IX71 and Cool SNAP HQcharge-coupled device camera (Roper Scientific JAPAN, Tokyo,Japan). Digital images were analyzed with Metamorph software(Universal Imaging, Downingtown, Pa.).

Effect of MG132 on stability of the HCV core protein. To determine the effect of proteasome inhibitors on the degradationof HCV core proteins in the presence of overexpressed PA28 ${gamma}$ ,2 x 10⁵ 293T cells were transfected with expression plasmidsencoding the HCV core protein and PA28 ${gamma}$ by lipofection on 35-mm-diameterplates. The proteasome inhibitor MG132 (Sigma) or solvent, dimethylsulfoxide, was added as a 10^-3 volume of medium into wells at8 h posttransfection. Cells were harvested at 24 h posttransfectionand lysed in lysis buffer as described above. Proteins weredetected by Western blotting with mouse monoclonal anti-Flag,anti-HA, goat polyclonal anti-actin (Santa Cruz Biotechnology,Santa Cruz, Calif.) or rabbit polyclonal anti-PA28 ${gamma}$ .

RESULTS

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Results

Isolation of PA28 ${gamma}$ cDNA from human libraries. To determine the protein(s) that interact with HCV core proteinin mammalian cells, we choose to employ a yeast two-hybrid systemwith the HCV core protein as bait. Human fetal brain and liverlibraries were used for this screening because it is not knownwhether the target protein is specifically expressed in theliver. Many light blue colonies emerged on dropout plates, butthese were eliminated from further screening so that proteinsexhibiting strong binding could be examined more fully. Severalclones exhibited dark blue color on a dropout plate containing5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside toan extent similar to that of the positive control containingp53 and large T antigen. No gene has been included which haspreviously been reported as a core-binding protein in the darkblue colonies, and we selected the darkest one. The total DNAwas extracted from this clone and introduced into E. coli strainJM109 with the goal of recovering the pACT2 plasmid encodingthe candidate core-binding protein. The nucleotide sequenceof the DNA insert was determined from three independent colonies.The sequence isolated from the positive clone included the 5'and 3' noncoding regions as well as the full coding region ofproteasome activator PA28 ${gamma}$ ; all sequences were in frame. Thereare two splicing variants of PA28 ${gamma}$ in human tissue (3, 23). Theisolated cDNA of PA28 ${gamma}$ encoded the major isoform that is comprisedof 254 amino acids; this isoform demonstrates 100% identitywith mouse PA28 ${gamma}$ based on amino acid sequence. The isolated pACT2plasmid containing PA28 ${gamma}$ cDNA was introduced into yeast strainAH109 together with either an empty bait plasmid, pGBKT7, ora plasmid encoding the HCV core protein, pGBKT7HCVCore173, inorder to confirm that the isolated plasmid encodes an HCV core-bindingprotein. The yeast clone containing pACT2-PA28 ${gamma}$ and pGBKT7HCVCore173grew on a dropout plate deficient in leucine, tryptophan, histidine,and adenine, but the yeast clone containing pACT2-PA28 ${gamma}$ and pGBKT7did not (data not shown). These data suggest that PA28 ${gamma}$ bindsto the HCV core protein in yeast. The cDNAs of HCV core proteinand its mutants were introduced into several mammalian expressionvectors as shown in Fig. 1.

Interaction of the HCV core protein with PA28 ${gamma}$ in mammalian cells, livers of HCV core transgenic mice, and a patient with chronic hepatitis C. Because it is generally known that many false-positive clonesare identified by using the yeast two-hybrid system, protein-proteininteraction and coincidence of intracellular localization betweenbait and prey proteins should be examined in mammalian cells.When Flag-tagged PA28 ${gamma}$ (Flag-PA28 ${gamma}$ ) was coexpressed in 293T cellswith HA-Core191, HA-Core173, HA-Core151, HA-Bad, or HA-FKBP,Flag-PA28 ${gamma}$ was coprecipitated with HA-Core191, HA-Core173, andHA-Core151 but not with HA-Bad and HA-FKBP by mouse anti-HAantibody. The interaction of Flag-PA28 ${gamma}$ with HA-Bad and HA-FKBPwas not observed even though these constructs were expressedat a higher level than the HA-Core proteins (Fig. 2A). To eliminatethe possibility of an artificial interaction of the HCV coreprotein with PA28 ${gamma}$ due to overexpression, the association ofHCV core proteins with endogenous PA28 ${gamma}$ was examined. EndogenousPA28 ${gamma}$ was coprecipitated with HCV core proteins in HA-Core-expressing293T cells but not in nontransfected cell lysates (Fig. 2B).

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FIG. 2. Interaction of HCV core protein with PA28 ${gamma}$ . (A) Human embryonic kidney 293T cells were transfected with the expression plasmids encoding HCV core proteins and/or PA28 ${gamma}$ . A Flag epitope tag was added to PA28 ${gamma}$ at its amino terminus while an HA epitope tag was added to the HCV core protein and control proteins (Bad and FKBP38). PA28 ${gamma}$ was able to be coimmunoprecipitated with the HCV core protein by anti-HA antibody and was visualized by Western blotting with anti-Flag antibody. HA-Core 151 was expressed by using two times amount of plasmid DNA compared to HA-Core 191 and 173 because coexpression with PA28 ${gamma}$ decreased the amount of HA-Core 151. (B) 293T cells were transfected with the expression plasmids encoding HA-tagged HCV core proteins. Endogenous PA28 ${gamma}$ was coimmunoprecipitated with HCV core proteins and was detected by immunoblotting with anti-PA28 ${gamma}$ antiserum. Asterisks indicate IgG bands. (C) Liver homogenates of HCV core transgenic mice and nontransgenic mice were immunoprecipitated (IP) with anti-core antibody; endogenous PA28 ${gamma}$ was coprecipitated with HCV core protein and was detected by immunoblotting with anti-PA28 ${gamma}$ antibody. (D) Liver homogenates of non-B and non-C hepatitis patients (lanes 1 and 2) and a patient with chronic hepatitis C (lane 3) were immunoprecipitated with anti-core antibody. Endogenous PA28 ${gamma}$ was coimmunoprecipitated with HCV core protein and was detected by immunoblotting with rabbit anti-PA28 ${gamma}$ antiserum.

Hepatic steatosis and hepatocellular carcinoma have been shownto be induced in transgenic mice expressing the HCV core protein;in this system, expression levels of the HCV core protein inmouse livers were similar to those in patients with chronichepatitis C (25, 39). The amino acid sequence of human PA28 ${gamma}$ is identical to that of mouse PA28 ${gamma}$ (22, 23, 44). Liver tissueof HCV core transgenic and nontransgenic mice were homogenizedin lysis buffer. Endogenous PA28 ${gamma}$ was coprecipitated with HCVcore protein by anti-HCV core antibody in liver lysates of HCVcore transgenic mice but not in those of nontransgenic mice(Fig. 2C), indicating that the HCV core protein specificallyinteracts with PA28 ${gamma}$ in the liver of core transgenic mice. Tofurther confirm the specific interaction of the HCV core proteinwith endogenous PA28 ${gamma}$ , this interaction was examined in liverspecimens from a patient with chronic hepatitis C infection(Fig. 2D). Endogenous PA28 ${gamma}$ was also coprecipitated with HCVcore protein in liver lysates from this patient (Fig. 2D, lane3), but not in patients with non-B and non-C hepatitis (Fig.2D, lanes 1 and 2), by anti-HCV core antibody. These resultsindicate that the HCV core protein specifically binds to PA28 ${gamma}$ not only in mammalian cell lines but also in liver tissue.

Intracellular localization of the HCV core protein with PA28 ${alpha}$ , ß, and ${gamma}$ . The nonessential, flexible loop region of PA28, termed the homologue-specificinsert region, lies between the N terminus of the protein andthe proteasome activation domain. This region does not showany homology with other PA28 isotypes (49). The nuclear localizationsignal (NLS) of PA28 ${gamma}$ encompasses amino acids 82 to 90 and lieswithin the homologous specific insert (amino acids 72 to 102);there is no NLS in PA28 ${alpha}$ and ß. PA28 ${gamma}$ is primarily localizedto the nucleus in mammalian cells through its NLS motif, butPA28 ${alpha}$ and ß are predominantly found in the cytoplasm(6). Figure 3 shows the intracellular localization of the HCVcore protein and endogenous PA28 ${gamma}$ and PA28 ${alpha}$ . HA-Core191 was predominantlydetected in the cytoplasm and to a lesser extent in the nucleusor perinuclear region in HeLa cells. Conversely, HA-Core173and HA-Core151 were primarily found in the nucleus with lesscytoplasmic staining. Endogenous PA28 ${gamma}$ was visualized by indirectimmunostaining with polyclonal rabbit anti-PA28 ${gamma}$ antiserum andwas predominantly detected in the nucleus of HeLa cells irrespectiveof the expression of HCV core proteins. HA-Core191 was partiallycolocalized with PA28 ${gamma}$ in the nucleus. In contrast to these findings,a large proportion of HA-Core151 or 173 was found to be colocalizedwith PA28 ${gamma}$ in the nucleus. PA28 ${alpha}$ and ß share 41.3 and33.6% homology to PA28 ${gamma}$ , respectively. A heteroheptamer of PA28 ${alpha}$ and ß binds to the 20S proteasome in the cytoplasmto activate the peptidase activity of this proteasome (1). EndogenousPA28 ${alpha}$ was predominantly detected in the cytoplasm and, to a lesserextent, in the nucleus. When HA-Core191 was expressed in HeLacells, it was mainly localized to the cytoplasm, but it didnot colocalize with PA28 ${alpha}$ . When HA-Core151 and 173 were expressedin HeLa cells, endogenous PA28 ${alpha}$ was not translocated from thecytoplasm to the nucleus, and no colocalization with HCV coreproteins was observed. Similar results were also obtained in293T cells (data not shown). Endogenous PA28 ${alpha}$ was not able tobe coimmunoprecipitated with Flag-HCV Core191 in 293T cells.Endogenous PA28 ${gamma}$ , however, was clearly coprecipitated with thecore protein (Fig. 4). Endogenous PA28ß was not colocalizedwith HCV core proteins in HeLa cells by indirect immunostaining(data not shown). These data indicate that the HCV core proteininteracts with PA28 ${gamma}$ but not with PA28 ${alpha}$ and ß.

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FIG. 3. Intracellular localization of HCV core protein with PA28 ${alpha}$ and ${gamma}$ . HeLa cells were transfected with plasmids encoding HA-Core proteins and then fixed with paraformaldehyde. The HCV core protein was visualized by indirect immunostaining with anti-HA antibody. Endogenous PA28 ${gamma}$ and ${alpha}$ were visualized by indirect immunostaining with rabbit anti-PA28 ${gamma}$ (A) and anti-PA28 ${alpha}$ (B) antisera, respectively. All samples were observed with a confocal microscope.

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FIG. 4. HCV core protein does not bind to PA28 ${alpha}$ . After overnight cultivation, 293T cells were transfected with the expression plasmid encoding Flag-HCV Core191. Immunoprecipitation (IP) was performed as described in Materials and Methods. Endogenous PA28 ${alpha}$ and PA28 ${gamma}$ coprecipitated with HCV core protein were stained with anti-PA28 ${alpha}$ and anti-PA28 ${gamma}$ antisera, respectively. Lc, light chain.

Intracellular localization of Flaviviridae core proteins with PA28 ${gamma}$ . The interaction of the HCV core protein with PA28 ${gamma}$ was demonstratedby coimmunoprecipitation, and the colocalization of these proteinswas examined by immunostaining. It was still unknown, however,whether the HCV core protein interacts with PA28 ${gamma}$ under livingcell conditions. Since the nuclear localization of PA28 ${gamma}$ is dependenton a c-Myc-like NLS, deletion of the NLS in PA28 ${gamma}$ should shiftits localization into the cytoplasm. When PA28 ${gamma}$ was fused tothe C terminus of the red fluorescence protein (DsRed) (DsRed-PA28 ${gamma}$ )and coexpressed with EGFP-Core151 in HeLa cells (Fig. 1), EGFP-Core151colocalized with DsRed-PA28 ${gamma}$ in the nucleus (Fig. 5A, upper panels).In the presence of DsRed-PA28 ${gamma}$ lacking the NLS (DsRed-PA28 ${gamma}$ ${Delta}$ NLS),however, EGFP-Core151 was predominantly detected in the cytoplasmand was colocalized with DsRed-PA28 ${gamma}$ ${Delta}$ NLS (Fig. 5A, lower panels).The detection of EGFP-Core151 in the nucleus of cells overexpressingDsRed-PA28 ${gamma}$ ${Delta}$ NLS was probably due to the interaction of the coreprotein with endogenous PA28 ${gamma}$ in the nucleus. The cytoplasmiclocalization of EGFP-Core151 was also detected with DsRed-PA28 ${gamma}$ ${Delta}$ NLSin 293T cells (data not shown). These data indicate that theHCV core protein binds to PA28 ${gamma}$ in living cells.

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FIG. 5. Interaction of Flaviviridae core proteins with PA28 ${gamma}$ . HeLa cells were transfected with the expression plasmids encoding DsRed-PA28 ${gamma}$ (upper panels) or DsRed-PA28 ${gamma}$ ${Delta}$ NLS (lower panels) together with EGFP-Core151 (A) or EGFP-JEV C (B). All samples were observed with a confocal microscope. (C) The cells were transfected with the expression plasmid encoding EGFP-Core151 (lane 1), EGFP-DEN C (lane 2), or EGFP-JEV C (lane 3) and then harvested at 36 h posttransfection. EGFP fusion proteins were precipitated with anti-GFP antibody. Endogenous PA28 ${gamma}$ was coimmunoprecipitated with anti-GFP antibody and then was visualized by immunoblotting with anti-PA28 ${gamma}$ antiserum. The untransfected cells were used as a negative control (lane 4). IP, immunoprecipitation; Hc, heavy chain; Lc, light chain.

DEN and JEV are both members of the Flaviviridae family, whichalso includes HCV (35, 50). The HCV core protein shares 22 and30% homology with the DEN and JEV core proteins within the N-terminal50 amino acids, respectively. Also similar to HCV, the coreproteins of DEN and JEV are basic. The EGFP-fused JEV core proteinlacking the C-terminal hydrophobic region (EGFP-JEV C) can bevisualized in both the cytoplasm and nucleus (Fig. 5B, upperpanels). The intracellular localization of EGFP-JEV C was quitedistinct from that of DsRed-PA28 ${gamma}$ , and coexpression with DsRed-PA28 ${gamma}$ ${Delta}$ NLSdid not affect the subcellular localization of the protein (Fig.5B, lower panels). Similar results were obtained by coexpressionof the EGFP-fused DEN core protein lacking the C-terminal hydrophobicregion (EGFP-DEN C). EGFP-DEN C was not colocalized with DsRed-PA28 ${gamma}$ and was not affected by expression of DsRed-PA28 ${gamma}$ ${Delta}$ NLS (data notshown). Endogenous PA28 ${gamma}$ was coprecipitated with EGFP-Core151by anti-GFP antibody but not with EGFP-DEN C or EGFP-JEV C (Fig.5C). These data suggest that PA28 ${gamma}$ specifically interacts withthe HCV core protein but not with DEN and JEV core proteinsin living cells.

Mapping of the PA28 ${gamma}$ -binding region of the HCV core protein. To determine the region of the HCV core protein responsiblefor PA28 ${gamma}$ binding, the interactions of PA28 ${gamma}$ with deletion mutantsof the HCV core protein were examined. When Flag-Core mutants(Fig. 1) were expressed in 293T cells, endogenous PA28 ${gamma}$ was coimmunoprecipitatedwith Flag-Core191, Flag-Core24-191, and Flag-Core38-191 by anti-Flagantibody but not with Flag-Core72-191 and Flag-Core92-191; thelevels of protein expression were the same for all constructs(Fig. 6A). Conversely, Flag-Core191, Flag-Core24-191, and Flag-Core38-191,but not Flag-Core72-191 and Flag-Core92-191, were coprecipitatedwith endogenous PA28 ${gamma}$ by anti-PA28 ${gamma}$ antibody. These results indicatethat the N-terminal 37 amino acids of the HCV core protein arenot involved in the interaction with PA28 ${gamma}$ . Because HA-Core151was shown to interact with PA28 ${gamma}$ (Fig. 2A) and localized to thenucleus (Fig. 3A), we examined the effect of deletion the N-terminalamino acids on the localization of Core-151 in living cellsby using EGFP-Core151 (Fig. 5A). EGFP-Core24-151 and EGFP-Core38-151were localized entirely within the nucleus, and EGFP-Core72-151and EGFP-Core92-151 were predominantly localized in the cytoplasm(Fig. 6B). These results give rise to the question of whetheramino acids 38 to 71 of the HCV core protein might be involvedin the interaction with PA28 ${gamma}$ and in the nuclear localizationof the HCV core protein. To determine the precise region ofthe HCV core protein responsible for binding with PA28 ${gamma}$ , we constructedadditional mutant core proteins, EGFP-Core38-43 and EGFP-Core44-71(Fig. 1). EGFP-Core44-71 was primarily localized to the nucleus,but EGFP-Core38-43 displayed a diffuse cellular staining similarto that of EGFP alone (Fig. 6B). EGFP-Core44-71, but not EGFP-Core38-43,was coprecipitated with endogenous PA28 ${gamma}$ by rabbit anti-GFP antiserumin 293T cells (Fig. 6C). These results suggest that a clusterof amino acids from 44 to 71 in the HCV core protein is responsiblefor both its interaction with PA28 ${gamma}$ and its nuclear localization.

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FIG. 6. Mapping of the PA28 ${gamma}$ -binding region in HCV core protein. (A) 293T cells were transfected with plasmids encoding Flag-tagged HCV core deletion mutants. Endogenous PA28 ${gamma}$ was coimmunoprecipitated with an anti-Flag antibody and was visualized by immunoblotting with anti-PA28 ${gamma}$ antiserum. (B) Intracellular localization of EGFP-Core deletion mutants in 293T cells. All samples were observed at 24 h posttransfection. (C) EGFP-tagged HCV core deletion mutants were expressed in 293T cells. Endogenous PA28 ${gamma}$ was coimmunoprecipitated with anti-GFP antiserum and then was visualized by immunoblotting with anti-PA28 ${gamma}$ antiserum. IP, immunoprecipitation.

Deletion of the PA28 ${gamma}$ -binding region or knockout of PA28 ${gamma}$ leads to export of the HCV core protein from nucleus to cytoplasm. To determine whether the PA28 ${gamma}$ -binding region identified in HCVcore protein amino acids 44 to 71 functioned as anNLS, the localizationof a deletion mutant lacking amino acids 44 to 71 was determined(Fig. 7A). EGFP-Core151 was detected in the nucleus of HeLacells and retained there until at least 48 h posttransfection.Conversely, EGFP-Core151 ${Delta}$ 44-71 (Fig. 1) was detected in the nucleusat 3 h posttransfection and gradually translocated into thecytoplasm. Most of the EGFP-Core151 ${Delta}$ 44-71 was detected in thecytoplasm at 24 h posttransfection. These results indicate thatHCV core protein amino acids 44 to 71 have a function in bothPA28 ${gamma}$ binding and nuclear retention. To further confirm thisobservation, we examined embryonic fibroblasts derived fromPA28 ${gamma}$ knockout mice (2) (Fig. 7B). When EGFP-Core151 was expressedin PA28 ${gamma}$ ^+/-or PA28 ${gamma}$ ^-/-mouse embryonic fibroblasts, EGFP-Core151was localized to the nucleus at 24 h posttransfection, irrespectiveof PA28 ${gamma}$ expression. EGFP-Core151 was retained in the nucleusof PA28 ${gamma}$ ^+/-mouse embryonic fibroblasts until 42 h posttransfection,when cell death was induced (Fig. 7B, left panels). In PA28 ${gamma}$ ^-/-fibroblasts,however, EGFP-Core151 was exported to the cytoplasm at 27 hposttransfection and no cell damage was observed until 44 hposttransfection (Fig. 7B, right panels). These data clearlyindicate that an interaction with PA28 ${gamma}$ is essential for thenuclear retention of the HCV core protein.

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FIG. 7. Intracellular localization of HCV core protein. (A) EGFP-Core151 and EGFP-Core151 ${Delta}$ 44-71 were expressed in HeLa cells. The intracellular localization of EGFP-Core151 (upper panels) or EGFP-Core151 ${Delta}$ 44-71 (lower panels) was observed at 3, 5, 10, and 24 h posttransfection. (B) Intracellular localization of EGFP-Core 151 in embryonic fibroblasts prepared from a PA28 ${gamma}$ ^+/- mouse (left) or from a PA28 ${gamma}$ ^-/- mouse (right) was examined from 24 to 42 h posttransfection by time-lapse microscopy.

Degradation of HCV core protein via PA28 ${gamma}$ -dependent pathway. It was previously reported that HCV core proteins truncatedat the C termini (HCV Core151 and 173), although normally rapidlydegraded, were able to be detected after the addition of a proteasomeinhibitor (57). To determine the effect of PA28 ${gamma}$ expression onthe stability of HCV core protein, HA-Core191, HA-Core173, orHA-Core151 was coexpressed with Flag-PA28 ${gamma}$ in 293T cells. Theamounts of HA-Core173 and HA-Core151 were decreased by overexpressionof Flag-PA28 ${gamma}$ , but expression levels of HA-Core191 were unchanged(Fig. 8A). Degradation of HA-Core151 by PA28 ${gamma}$ overexpressionwas eliminated by the addition of the proteasome inhibitor MG132(Fig. 8B), thus suggesting that nucleus-localized HCV core proteinundergoes degradation by the proteasome in a PA28 ${gamma}$ -dependentmanner. To confirm the nuclear localization and degradationof the processed HCV core proteins derived from HA-Core191,MG132 was added to HeLa cells transfected with the plasmid encodingHA-Core191 (Fig. 8C). Treatment with MG132 enhanced the expressionof HCV core protein colocalized with endogenous PA28 ${gamma}$ in thenucleus of HeLa cells expressing HA-Core191. F protein was generatedby the -2/+1 ribosomal frameshift in the gene encoding HCV coreprotein (64). The expected molecular mass of the F protein ofthe J1 strain is about 14 kDa. Endogenous PA28 ${gamma}$ was coprecipitatedby anti-Flag antibody with Flag-Core151 but not with Flag-Fprotein (Fig. 9). These results suggest that the HCV core proteinis processed by the cleavage of the C-terminal hydrophobic regionand that the truncated core protein or the mature protein istranslocated into the nucleus and degraded in a PA28 ${gamma}$ -dependentmanner.

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FIG. 8. The proteasome inhibitor MG132 blocks degradation of the HCV core protein. (A) 293T cells were transfected with the expression plasmids encoding HA-Core 191, 173, or 151 together with an empty plasmid or a plasmid encoding Flag-PA28 ${gamma}$ . Cell lysates were analyzed by immunoblotting with anti-HA, anti-Flag, or anti-actin antibodies. (B) 293T cells were transfected with an expression plasmid encoding HA-Core 151 with either an empty plasmid or a plasmid encoding Flag-PA28 ${gamma}$ . Cells were treated with either MG132 in dimethyl sulfoxide (DMSO) or dimethyl sulfoxide alone as a control where indicated. (C) HeLa cells transfected with plasmid encoding HA-Core191 were treated with 30 µM MG132 at 10 h posttransfection and then fixed with paraformaldehyde at 24 h posttransfection. Endogenous PA28 ${gamma}$ and HA-Core were stained with rabbit anti-PA28 ${gamma}$ antiserum and mouse anti-HA antibody, respectively. All samples were observed with a confocal microscope. +, present; -, absent.

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FIG. 9. The F protein of the -2/+1 frame does not bind to PA28 ${gamma}$ . The plasmid encoding Flag-Core151 (lane 2) or Flag-F (lane 3) protein was transfected into 293T cells and harvested at 36 h posttransfection. Endogenous PA28 ${gamma}$ was coimmunoprecipitated with anti-Flag antibody and then was visualized by immunoblotting with anti-PA28 ${gamma}$ antiserum. The cells transfected with the empty plasmid were used as a negative control (lane 1). IP, immunoprecipitation; Lc, light chain.

DISCUSSION

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Discussion

The mechanism of hepatocellular carcinoma development in patientswith chronic hepatitis C remains unclear. It has been demonstratedthat expression of the HCV core protein alone is sufficientfor the induction of hepatic steatosis and hepatocellular carcinomain transgenic mice (28, 39, 41). These findings suggest thatthe HCV core protein plays a pivotal role in the developmentof hepatocellular carcinoma. In this study, we isolated PA28 ${gamma}$ from a human fetal brain library as a host protein that specificallybinds to the HCV core protein. We further suggest that HCV coreprotein interaction with PA28 ${gamma}$ correlates with the retentionof HCV core protein in the nucleus and regulates the stabilityof the HCV core protein in a proteasome-dependent manner. Thereare two isoforms of PA28 ${gamma}$ in humans, a major form and a splicingvariant that contains an additional 13 amino acids in the secondhelix domain. The second isoform is detected only in the humanfetal brain and is not found in other human tissues or othermammals (3, 23). In this screen, we did not obtain the splicingvariant of PA28 ${gamma}$ from the human fetal brain library; it is, therefore,still unknown whether the human-specific isoform of PA28 ${gamma}$ bindsto the HCV core protein.

The C-terminal hydrophobic region of the HCV core protein isprocessed by host proteases such as signal peptidase and/orintramembrane proteases. The processed, mature HCV core proteintransferred into lipid droplets when a full length of core proteinwas expressed by an alphavirus expression system (14, 27, 36).However, the mature core protein remained in the ER when thefull length of core protein was expressed by transfection inthis study (Fig. 3). This discrepancy might be due to the differencein expression systems, cell lines, and genotypes of the HCVclone.

When fused to EGFP, the PA28 ${gamma}$ -binding region of the HCV coreprotein (EGFP-Core44-71) migrated into the nucleus, indicatingthat this region may function as an NLS. Deletion of the PA28 ${gamma}$ -bindingregion from the HCV core protein (EGFP-Core151 ${Delta}$ 44-71) or depletionof PA28 ${gamma}$ from cells, however, did not eliminate nuclear transportof the HCV core protein, suggesting the presence of an alternativemechanism for the nuclear transport of the HCV core proteinother than its interaction with PA28 ${gamma}$ . Within the C-terminallytruncated HCV core protein there exist three putative NLSs consistingof a cluster of basic amino acids (8, 55). ß-Galactosidase-fusedC-terminal truncated core protein lacking one of these clusters(ß-gal-Core123 ${Delta}$ 38-43) was localized primarily in thecytoplasm rather than the nucleus in COS cells (55); an EGFP-fusedmutant, EGFP-Core151 ${Delta}$ 38-43, however, was localized in the nucleusin the HeLa and 293T cell lines (data not shown). These resultssuggest that there are at least two possible mechanisms, PA28 ${gamma}$ dependent and PA28 ${gamma}$ independent, leading to nuclear transportof the HCV core protein. EGFP-Core151 ${Delta}$ 38-43 and EGFP-Core151 ${Delta}$ 44-71are translocated into the nucleus by the PA28 ${gamma}$ -dependent and-independent pathways, respectively. Both pathways may be mediatedthrough importin or importin-like molecules because PA28 ${gamma}$ hasa c-Myc-like NLS in its homolog-specific region. Furthermore,the interaction with PA28 ${gamma}$ was shown by time-lapse microscopyto play an important role in the retention of the HCV core proteinin the nucleus. HCV core proteins lacking the PA28 ${gamma}$ -binding region,EGFP-Core151 ${Delta}$ 44-71 and EGFP-Core151, were exported from the nucleusto the cytoplasm in HeLa cells and embryonic fibroblasts derivedfrom PA28 ${gamma}$ knockout mice, respectively. The nuclear exportingsignal was found in the C-terminal half of the HCV core proteinand plays a role in the export of the HCV core protein fromthe nucleus to the cytoplasm (R. Suzuki, S. Sakamoto, T. Tsutsumi,A. Rikimaru, T. Shimoike, S. Machida, Y. Matsuura, T. Miyamura,and T. Suzuki, unpublished data). The putative PA28 ${gamma}$ -dependentand -independent translocation of the HCV core protein fromthe cytoplasm to the nucleus, as well as the possible functionsand fates of the HCV core protein in the nucleus, are illustratedin Fig. 10.

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FIG. 10. Putative PA28 ${gamma}$ -dependent and -independent translocation of the HCV core protein from the cytoplasm into the nucleus and possible function and fate. A precursor HCV core protein (Core191) is processed from a polyprotein in the ER by signal peptidase (red triangle), and then signal peptide peptidase (blue triangle) cleaves the C-terminal hydrophobic region, resulting in generation of mature core protein (Core173). The C-terminal region of the mature core protein is further processed by an unknown host protease (gray triangle) (Core151). Core173 and Core151 migrate into the nucleus through the nuclear pore complex, presumably by importin or an importin-PA28 ${gamma}$ complex. HCV core proteins interacting with PA28 ${gamma}$ in the nucleus are degraded by the proteasome. This proteolysis is inhibited by the addition of the proteasome inhibitor MG132. Nuclear localization and degradation of the HCV core protein may induce down regulation of viral assembly, which may contribute to the maintenance of persistent infection with HCV. Furthermore,the resulting HCV core polypeptides might have some role as transcriptional regulators of host genomes, which are involved in hepatocarcinogenesis.

Although many host proteins have been reported to interact withthe HCV core protein in relation to carcinogenesis (18, 33,46, 66, 67), this is the first report demonstrating the interactionof the HCV core protein with an endogenously expressed hostprotein. In the livers of HCV core transgenic mice, the HCVcore protein was primarily detected in the cytoplasm but someprotein was found in the nucleus, albeit to a lesser extent(40). PA28 ${gamma}$ was shown to coimmunoprecipitate with HCV core proteinsirrespective of their intracellular localization (Fig. 2 and3), suggesting that the core proteins bind to PA28 ${gamma}$ after celldisruption. HCV core proteins truncated at the C terminus (HCVCore151 and 173) migrated into the nucleus and were degradedby ubiquitin-mediated proteolysis (57). In this study, overexpressionof PA28 ${gamma}$ led to the degradation of the HCV core protein; thisdegradation was able to be partially blocked by the proteasomeinhibitor MG132. Additionally, HCV core protein was detectedin the nucleus of a HeLa cell expressing the full-length HCVcore protein in the presence of MG132 (Fig. 8). These resultssuggest that the HCV core protein migrates into the nucleusand is then promptly degraded by the nuclear proteasome.

The F protein generated by ribosomal frameshift in the geneencoding the core protein was mainly localized in the cytoplasmand degraded by the proteasome (63). Although the expected massof 14 kDa of the F protein from strain J1 was not detected inHeLa cells expressing HA-Core151 even in the presence of MG132(Fig. 8B), we examined the interaction of the protein of -2/+1frame of the gene encoding the HCV core protein with PA28 ${gamma}$ . Lackof interaction of endogenous PA28 ${gamma}$ with the F protein (Fig. 9)suggests that PA28 ${gamma}$ specifically interacts with the HCV coreprotein but not with the F protein.

Hepatitis B virus X factor (HBx) alone induces hepatocellularcarcinoma in mice (20, 24), suggesting that HBx plays an importantrole in hepatocellular carcinoma. HBx bound to PSMA7 and PSMC1,subunits of PA700 and the 20S proteasome, respectively, leadsto the enhancement of the transcription activities of AP-1 andVP-16 (69). Like HBx, the HCV core protein is processed by theproteasome in a PA28 ${gamma}$ -dependent manner. An HCV core protein withthe same molecular mass as HCV Core151 was detected in cellsin the presence of MG132 (57). The proteasome is well knownto regulate many transcription factors such as NF- ${kappa}$ B, p53, andc-Myc, etc. (4). For example, NF- ${kappa}$ B and its inhibitor I ${kappa}$ B aredegraded by the proteasome, resulting in translocation of activeNF- ${kappa}$ B into the nucleus (19). Upon processing, the active formof NF- ${kappa}$ B acquires transcription activity that regulates manybiological functions such as cell proliferation (43). The HCVcore protein is known as a regulatory factor that modulatessome signaling pathways as well as affecting expression levelsof a variety of proteins under the control of different promoters(reviewed in reference 56). The short-lived, C-terminally truncatedHCV core protein may acquire an as yet undetermined biologicalfunction in the nucleus. Additionally, peptides derived fromthe HCV core protein that has been processed by the PA28 ${gamma}$ -activatedproteasome may play some role in the transcriptional regulationthat is involved in hepatocellular carcinogenesis.

The PA28 ${gamma}$ homopolymer is able to associate with the 20S proteasome(60) and strongly activates the peptidase activity of the latentproteasome (48). The PA28 ${alpha}$ /ß heteropolymer forms ahybrid proteasome with the 20S proteasome and PA700; this complexefficiently enhances antigen processing in an ATP-dependentmanner (59). The PA28 ${gamma}$ homopolymer, PA700, and the 20S proteasomemay also form a hybrid proteasome that may be responsible forthe proteolysis of the HCV core protein in the nucleus. PA28 ${gamma}$ knockout mice demonstrate no abnormality other than growth retardation;this suggests that PA28 ${gamma}$ is either dispensable for host physiologicalfunction or that suitable compensation mechanisms exist withinthe organism (42). Translocation and degradation of the HCVcore protein by the PA28 ${gamma}$ -activated proteasome in the nucleusmay also contribute to the establishment and maintenance ofpersistent infection of HCV through the down regulation of viralassembly.

Although the biological significance of PA28 ${gamma}$ is not well understood,in this study we have demonstrated new mechanisms by which PA28 ${gamma}$ translocates and retains the HCV core protein in the nucleus;PA28 ${gamma}$ is also involved in the proteolysis of the HCV core protein.Another nuclear proteasome activator, PA200, was recently purifiedfrom bovine testis and was demonstrated to enhance the peptidaseactivity but not the protease activity of the 20S proteasome(61). This report suggests that PA200 may be the functionalhomologue of PA28 in the nucleus. PA200 is predominantly localizedto the nucleus and demonstrates homology to yeast and worm proteinsthat are implicated in the repair of DNA double-strand breaks.Thus, nuclear proteasome activity may be associated with DNArepair. Therefore, it may be possible that the interaction ofPA28 ${gamma}$ with the HCV core protein results in a perturbation ofDNA repair activity through the nuclear proteasome, and thesechanges may subsequently induce hepatocellular carcinoma inhumans and mice.

In conclusion, we have demonstrated that PA28 ${gamma}$ specifically interactswith the HCV core protein in cell culture as well as in thelivers of both HCV core transgenic mice and a patient with chronichepatitis C. This interaction correlates to the nuclear retentionand degradation of C-terminally truncated HCV core proteins.Understanding the precise function of PA28 ${gamma}$ may give us new insightinto virus-cell interactions and lead to a greater understandingof the pathogenicity of HCV infection. Establishment of HCVcore transgenic mice deficient in PA28 ${gamma}$ gene expression willallow the direct assessment of the involvement of PA28 ${gamma}$ in thedevelopment of hepatocellular carcinoma induced by HCV coreprotein; these experiments are under way.

ACKNOWLEDGMENTS

We thank T. Shioda for advice on confocal microscopy, S. Ogawafor assistance in sequencing and the preparation of experimentalreagents, and T. Abe, K. Okamoto, and Y. Mori for critical readingof the manuscript.

This work was supported in part by grants-in-aid from the Ministryof Health, Labor, and Welfare, the program for Promotion ofFundamental Studies in Health Sciences of the Organization forDrug ADR Relief, R&D Promotion, and Product Review of Japanto Y.M. and T.M. and the Ministry of Education, Science, andCulture of Japan to Y.M.

FOOTNOTES

* Corresponding author. Mailing address: Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita-shi, Osaka 565-0871, Japan. Phone: 81-6-6879-8340. Fax: 81-6-6879-8269. E-mail: matsuura{at}biken.osaka-u.ac.jp.

REFERENCES

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