Interaction of the Hepatitis B Virus X Protein with theCrm1-dependent Nuclear Export Pathway*

Received for publication, February 8, 2001, and in revised form, March 29, 2001Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101259200

Marshonna Forgues‡, Aizen J. Marrogi‡, Elisa A. Spillare‡, Chuan-Ging Wu‡, Qin Yang‡,Minoru Yoshida§, and Xin Wei Wang‡¶

From the ‡Laboratory of Human Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892 and the§Department of Biotechnology, The University of Tokyo, Tokyo, Japan

The leucine-rich nuclear export signal (NES) is used toshuttle large cellular proteins from the nucleus to thecytoplasm. The nuclear export receptor Crm1 is essen-tial in this process by recognizing the NES motif. Here,we show that the oncogenic hepatitis B virus (HBV) Xprotein (HBx) contains a functional NES motif. Wefound that the predominant cytoplasmic localization ofHBx is sensitive to the drug leptomycin B (LMB), whichspecifically inactivates Crm1. Mutations at the two con-served leucine residues to alanine at the NES motif(L98A,L100A) resulted in a nuclear redistribution ofHBx. A recombinant HBx protein binds to Crm1 in vitro.In addition, ectopic expression of HBx sequesters Crm1in the cytoplasm. Furthermore, HBx activates NFkB byinducing its nuclear translocation in a NES-dependentmanner. Abnormal cytoplasmic sequestration of Crm1,accompanied by a nuclear localization of NFkB, was alsoobserved in hepatocytes from HBV-positive liver sam-ples with chronic active hepatitis. We suggest that Crm1may play a role in HBx-mediated liver carcinogenesis.

Hepatocellular carcinoma (HCC)1 is one of the most preva-lent malignant diseases worldwide and hepatitis B virus (HBV)is the major etiologic factor for HCC (1–3). HBV is a DNAtumor virus, which encodes four open reading frames, S/preS,C/preC, P, and HBx (4). The oncogenicity of HBV is largely theresult of HBx, the smallest gene encoding a 17-kDa protein (3,5–8). One major cellular function of HBx is its promiscuoustranscriptional activation activity, a property that is believedto contribute to its oncogenicity (9). A wide range of cellulargenes can be up-regulated or down-regulated by HBx (9). How-ever, HBx is localized in the cytoplasm mostly and does notbind to double-stranded DNA. A “universal” effect of HBx, onotherwise totally different types of promoters with no obviousconsensus sequence, has led to the hypothesis that HBx mayregulate gene expression by interacting directly with host gen-eral transcription factors (10–12), or indirectly via the activa-

tion of protein kinase C and RAS-RAF mitogen-activated pro-tein kinase (MAPK) signaling pathways (13–15). AlthoughHBx can induce neoplastic transformation, presumably by pre-venting p53-mediated apoptosis (16–18), it also can induceapoptosis in a p53-dependent or -independent manner (19, 20),2

or sensitize cells to tumor necrosis factor a (TNFa)-inducedapoptosis (21). Therefore, the precise mechanism related to itseffector remains unknown and none of these studies satisfac-torily explain the pleiotropic effects associated with HBx.

Close inspection of the HBx sequence revealed a short, hy-drophobic, leucine-rich nuclear export signal motif (NES) (Fig.1A). An NES is located in the center region of HBx (residues89–100). The center region of HBx is retained in HCC fre-quently and is essential for its transactivation (22–24). Thisregion also is conserved among HBx from different subtypes(Fig. 1A). Several viral proteins including HIV-1 Rev, HTLV-1Rex, and adenovirus E4 34-kDa proteins contain functionalNESs (Fig. 1A). Similar to HBx, Rev and Rex also are potentviral and cellular transactivators with no apparent DNA bind-ing property (25, 26). In addition, NESs also have been identi-fied in cellular proteins, many of which are involved in tran-scription, cell signaling cascade, oncogenic transformation, andcell cycle regulators. Examples include protein kinase inhibi-tor, MAP kinase kinase (MAPKK), TFIIIA, Mdm2, p53, IkBa,NF-AT, cyclin B1, c-Abl, and 14-3-3 (reviewed in Ref. 27). Theactivities of these proteins are tightly regulated by their NESs.The nuclear export receptor Crm1 and its cofactor Ran GTPaseare essential in this process by recognizing NESs and mediat-ing nuclear protein export (27, 28). In addition, previous resultsindicate that Crm1 may be involved in maintaining chromo-somal integrity (29) and Ran may play a key role in regulatingmitosis initiation by stimulating spindle formation (30, 31).Mutation of the hydrophobic leucine residues to alanines havebeen shown to disrupt NES function in a number of proteins,including HIV-rev, E4 34-kDa, p53, Mdm2, and cyclin B1 (25,26, 32, 33).

In this study, we have investigated the hypothesis that thepleiotropic effects associated with HBx may be contributed bythe presence of a NES motif, and HBx may activate cellulargene expression and induce oncogenicity through the modula-tion of Crm1-mediated functions. We have identified a func-tional NES motif in HBx. This motif is necessary for HBx-induced cytoplasmic sequestration of Crm1, and subsequently,the nuclear translocation and activation of NFkB. Cytoplasmicretention of Crm1 also is found in liver samples with chronicactive hepatitis infected with HBV, a condition that is predis-posing individuals to the development of HCC. We suggest that

* This work was supported by the Intramural Research Program ofthe National Cancer Institute. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

¶ To whom reprint requests should be addressed: LHC, NCI, NationalInstitutes of Health, 37 Convent Dr., MSC 4255, Bldg. 37, Rm. 2C07,Bethesda, MD 20892-4255. E-mail: xin_wei_wang@nih.gov.

1 The abbreviations used are: HCC, hepatocellular carcinoma; NES,nuclear export signal; NLS, nuclear localization signal; HBx, hepatitisB viral X protein; HBV, hepatitis B virus; NHF, normal human fibro-blasts; LMB, leptomycin B; MAPK, mitogen-activated protein kinase;TNFa, tumor necrosis factor-a; HA, hemagglutinin; PCR, polymerasechain reaction; GFP, green fluorescent protein; GTPgS, guanosine59-3-O-(thio)triphosphate.

2 M. Forgues, A. J. Marrogi, E. A. Spillare, C-G. Wu, Q. Yang, M.Yoshida, and X. W. Wang, unpublished data.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 25, Issue of June 22, pp. 22797–22803, 2001Printed in U.S.A.

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the inactivation of the Crm1-mediated pathway may be anearly step during viral hepatitis-mediated liver carcinogenesis.

EXPERIMENTAL PROCEDURES

Plasmid Construction—The plasmid pcDNA3-HBxadr-Hatag wasconstructed by the insertion of a C-terminal hemagglutinin (HA)epitope-tagged full-length HBx into the BamHI and HindIII sites of apcDNA3 vector (Invitrogen). GFP-HBx and GFP-HBx-NESM were con-structed as follows: a 529-base pair fragment released from the diges-tion of pcDNA3-HBxadr-Hatag with HindIII/ApaI and inserted intopEGFP-C2 (CLONTECH) at the HindIII/ApaI sites. The resulting GFP-HBx is an N-terminal fusion of HBx with GFP and a C-terminal fusionwith HA tag. GFP-HBx-NESM was made by a PCR-based site-directedmutagenesis protocol using GFP-HBx as a template. To generate theHBx-NES mutant (L98A,G99A,L100A), 2 HBx fragments were ampli-fied by PCR using primer pairs (59-GCTGCTGCATCAGCAATGTCAAC-AACCGAC-39/59-ATGCTCTAGAGGCAGAGGTGAAAAAGTTGCATG-G-39 and 59-TGCAGCAGCAGTCCTCTTATGTAAGAGCTT-39/59-TATA-AAGCTTGGTACCGAGCTCGGATCTGATGGC-39). The 2 PCR DNAfragments were denatured and reannealed. Then the HBx-NES mutantwas generated by PCR with primers (59-TATAAAGCTTGGTACCGAG-CTCGGATCTGATGGC-39 and 59-ATGCTCTAGAGGCAGAGGTGAAA-AAGTTGCATGG-39) and was subcloned into the pEGFP-C2 vector.Mutations were verified by DNA sequencing. The GFP-NLS-NES andGFP-NLS-NESM constructs were made by the strategy describedpreviously (34). Briefly, the SV40 NLS motif (residues 125–133) wasfused to GFP by the cloning of oligos TNLS1 (59-TCGAGATCCCCCC-AAGAAGAAGCGCAAGGTGGAGCA-39/TNLS2 59-AGCTTGCTCCAC-CTTGCGCTTCTTCTTGGGGGGATC-39) into plasmid pGFPF-C1(CLONTECH) to generate GFP-NLS. GFP-NES and GFP-NESM wereconstructed by cloning oligos XNESW1 (59-AATTCTCAGGTCTTGCCCA-AGCTCTTACATAAGAGGACTCTTGGACTCTCAGCAATG-39)/XNESW2(59-GATCCATTGCTGAGAGTCCAAGAGTCCTCTTATGTAAGAGCTT-GGGCAAGACCTGAG-39) or XNESM3 (59-AATTCTCAGGTCTTGCCC-AAGCTCTTACATAAGAGGACTGCTGGAGCCTCAGCAATG-39)-/XNESM4 (59-GATCCATTGCTGAGGCTCCAGCAGTCCTCTTATGTA-AGAGCTTGGGCAAGACCTGAG-39), respectively, into GFP-NLS,resulting in a GFP-NLS-NES or GFP-NLS-NESM fusion protein. TheNES motif corresponds to the HBx residues 87–103, while NESM is theHBx-NES motif with mutations at the conserved leucine residues(L98A and L100A). pSVCM contains a GFP-CRM1 fusion gene underthe control of a CMV promoter (28). pNFkB-Luc, provided by JohnBrady (NCI), contains a luciferase gene under the promoter containing4 NFkB responsive elements.

Cell Culture and Media—Normal primary human fibroblasts wereobtained from Coriell Institute for Medical Research (Camden, NJ).These cells were grown in Ham’s F-10 medium supplemented with 15%fetal bovine serum and were used before passage 12. A telomerase-immortalized human fibroblast line (NHF-hTERT), a gift of Dr. JudyCampisi, was grown in Dulbecco’s modified Eagle’s media supple-mented with 10% fetal bovine serum. Normal primary human hepato-cytes were purchased from Clonetics (BioWittaker), grown in LCM(BioWittaker), and used only at passage 1. Hep3B cells were grown inEagle’s minimum essential medium containing 10% fetal bovine serum.

Microinjection, Transfection, Luciferase, and Indirect Immunofluo-rescence Assays—Microinjection of NHF cells was done essentially asdescribed previously (35). Transfection of NHF-hTERT and Hep3B cellswas carried out by the LipofectAMINE Plus reagent based on therecommended protocol described by the manufacturer (Life Technolo-gies, Inc., Gaithersburg, MD). For the luciferase reporter assay, cellswere seeded into 6-well dishes (Costar) at 50% confluence, co-trans-fected with 0.2 mg of pNFkB-Luc or pNFAT-Luc, along with 2 mg of eachof various HBx constructs, in the presence or absence of TNFa (10ng/ml). A total of 0.5 mg of pRL-null or 0.003 mg of pRL-CMV was addedinto each transfection as an internal control. pEGFPC1 was used tokeep the total amount of plasmid DNA constant. Cells were then incu-bated for 24 h prior to harvesting. The luciferase activity was carriedout by the Dual Luciferase Reporter Assay System (Promega) accordingto the manufacturer’s instructions and were measured in a Monolight2010 luminometer (Analytical Luminescence Laboratory). The lucifer-ase activity was expressed as fold activation against the untreatedreporter by itself, which was normalized by Renilla luciferase activityfor the transfection efficiency. The reported results represent at leastthree independent transfections. For immunocytochemistry analysis,cells were fixed in 4% paraformaldehyde in phosphate-buffered salinefor 10 min, followed by methanol for 20 min at 24 h postmicroinjection,and washed in phosphate-buffered saline-plus (0.15% glycine, 0.5%

bovine serum albumin in phosphate-buffered saline). The GFP fusionproteins can be visualized directly in a fluorescence microscopeequipped with an fluorescein isothiocyanate filter with or without fix-ation. The HBx expression also was verified by an anti-HBx monoclonalantibody (data not shown). NFkB was detected using an anti-NFkBpolyclonal antibody (1:100) (Santa Cruz) and Crm1 was detected usingan anti-Crm1 polyclonal antibody (1:100). Both antibodies were incu-bated for 1 h at 37 °C. Anti-rabbit IgG conjugated to fluorescein orTexas Red (Vector Laboratories) was used (1:300) for 1 h at roomtemperature. Nuclei were stained with 4,6-diamidino-2-phenylindole.The following criteria were used to define the subcellular localization,preferentially cytoplasmic localization: protein signals mostly intensein the cytoplasm; preferentially nuclear localization: protein signalsmostly intense in the nucleus. Some of the slides were analyzed with ablind fashion to avoid any bias. For GFP-NLS reporter expression, cellson the same coverslip were microinjected with GFP-NLS, GFP-NLS-XNES, or GFP-NLS-XNESM construct and incubated for 6 h. Cellswere monitored under a Zeiss Axioskop fluorescence microscope andrepresentative images were taken using a high-performance CCD im-aging system (IP Lab Spectrum) with the same exposure time. Forco-localization of Crm1 and HBx analysis, cells were co-stained withanti-HBx and anti-Crm1 antibodies with the corresponding secondaryantibodies conjugated with Texas Red or fluorescein, and analyzed by aBio-Rad MRC 1024 confocal system. Sequential excitation at 488 and568 nm was provided by a krypton-argon gas laser. Emission filters of598/40 and 522/32 were used for sequentially collecting red and greenfluorescence in channels 1 and 2, respectively. Z-sections were collectedat 0.5-mm intervals for each cell using LaserSharp software. Imageswere analyzed by Confocal Assistant software.

In Vitro Protein-binding Assay—In vitro binding between HBx andhCrm1 was determined with the in vitro translated HBx and hCrm1(16). The HA-tagged HBx and the hCrm1 proteins were made by theone-step TnT in vitro transcription and translation system (Promega),as described previously (16), from pcDNA-HA-HBx and hCrm1Blue744vectors (gift of Dr. Gerard Grosveld), respectively. Aliquots of HBx (25ml) and hCrm1 (45 ml) were mixed together in CBBL buffer (50 mM

Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM

dithiothreitol, and 0.1% Nonidet P-40) in the presence of 0.2 mM GTPgSand 80 ml of anti-HA affinity matrix (Roche Molecular Biochemicals,Indianapolis, IN). The mixture was incubated at 37 °C for 60 min. Aftercentrifugation, beads were washed four times with CBBL buffer. Thebound proteins were separated on SDS-polyacrylamide gel electro-phoresis and Crm1 was detected by Western blotting using anti-Crm1antibody and followed by the Amersham ECL system. A 10% of in vitrotranslated hCrm1 was loaded in parallel as the input.

Immunohistochemistry Analysis—A total of 12 samples were in-cluded in this part of the study. Histopathologically, they included 5normal liver samples, 7 viral hepatitis cases showing grades 1 to 2activity according to the criteria of Scheuer (36). In addition to H & Elight microscopy, tissue sections were cut from frozen samples with athickness of 4 mm and mounted on electrically charged glass slides. Thesections were fixed in absolute alcohol and then washed with 2 changesof phosphate-buffered saline for 5 min each. They were then quenchedwith 3% hydrogen peroxide to block the endogenous peroxidase activityfor 30 min. Following incubation with 10% horse serum to block thenonspecific binding, the sections were incubated overnight at 4 °C withanti-CRM1 antibodies. The sections were prepared in duplicate and twoanti-CRM1 antibodies were used, including a goat anti-Crm1 antibodyfrom Santa Cruz at a 1:10 dilution (Santa Cruz) and a rabbit anti-Crm1polyclonal antibody (Gift from Gerard Grosveld) at a 1:100 dilution. Arabbit anti-NFkB antibody (anti-p65) (Santa Cruz) was used at a 1:100dilution for staining NFkB. Biotinylated secondary antibodies andstreptavidin peroxidase complex (LSAB) were used. Chromogenic de-velopment was obtained by the immersion of sections in 3,39-diamino-benzidine solution (0.25 mg/ml with 3% hydrogen peroxide). The slideswere counterstained with Harris’ hematoxylin and re-hydrated withalcohol and xylene. Slides were evaluated in a blind fashion.

RESULTS

HBx Has a Functional Leucine-rich Nuclear Export Sig-nal—To test if HBx-NES functions in nuclear protein export,we first investigated whether the subcellular localization ofHBx is sensitive to LMB, an antitumor agent that selectivelybinds to and blocks Crm1-mediated nuclear export (37, 38).Consistent with previous findings (18), HBx was localized inthe cytoplasm predominately with a punctate staining pattern

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in appearance when expressed transiently in normal humanfibroblasts (NHF) (Fig. 1) and in a HCC-derived cell line(Hep3B) (data not shown). However, LMB treatment causes anincrease in nuclear accumulation of HBx (Fig. 1, B and C).

Next, we used a GFP protein export reporter assay describedpreviously (34) to further test if the HBx-NES oligopeptidefunctions in nuclear protein export (34). This reporter uses anuclear localization signal (NLS) fused to a GFP, thereby in-

FIG. 1. The hepatitis B virus HBxprotein contains a functional NES. A,sequence comparison of the leucine-richNESs between 3 subtypes of HBx andother proteins with known NES function.Dots indicate the critical residues, inwhich mutations are known to inactivatethe activity of the NESs. B, effect of LMBon subcellular localization of HBx in pri-mary human fibroblasts. HBx was ex-pressed transiently in normal human fi-broblasts via microinjection. Following24 h incubation, cells were treated with(right panels) or without (left panels) 4 nM

LMB for 2 h. HBx protein was detected byindirect immunofluorescence stainingwith monoclonal anti-HBx antibody andfollowed by fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (toppanels). Nuclei of the same cells werestained with 4,6-diamidino-2-phenylin-dole (DAPI) (bottom panels). Representa-tive images are shown (magnification is 3600). C, percent of cells with cytoplasmiclocalization of HBx from B. D, HBx-NESis functional in nuclear export. SeveralGFP fusion constructs were expressedtransiently in normal primary human fi-broblasts. Living cells were monitored un-der a fluorescence microscope. A nuclearlocalization signal from SV40 T antigendirects GFP to the nucleus (left panel)while the HBx-NES redistributes it to thecytoplasm (middle panel). Mutations ofleucine to alanine in NES abolish thisactivity (right panel). E, the HBx-NESmutant is localized preferentially in thenucleus. Normal human fibroblasts weremicroinjected with the GFP-HBx or GFP-HBx-NESM expression vector. Followinga 24-h incubation, cells were fixed andstained with 4,6-diamidino-2-phenylin-dole. The percent of cells in which theGFP signal was found preferentially inthe cytoplasm (left panel), in the nucleus(middle panel), or both (evenly distribut-ed; right panel) was determined. Exa-mples of these images are shown. Dataare an average of three independentexperiments.

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ducing nuclear localization of the fusion protein. Consistently,the GFP-NLS reporter was localized in the nucleus of NHFexclusively. However, when an HBx-NES was fused to thisreporter, the resulting GFP-NLS-XNES fusion protein can befound in the cytoplasm with a diffused staining pattern inappearance, although no nuclear exclusion of this reporterprotein could be observed (Fig. 1D). In contrast, GFP-NLS-XNESM that contains a mutated HBx-NES oligopeptide(L98A,L100A) does not localize in the cytoplasm (Fig. 1D). Tofurther examine if cytoplasmic localization of a full-length HBxis dependent on the presence of an NES, a GFP-HBx fusiongene (HBx) or an NES-mutated GFP-HBx fusion gene (HBx-NESM) was constructed. Again, GFP-HBx was localized in thecytoplasm predominantly with a punctate pattern in appear-ance, while HBxNESM was found mainly in the nucleus with adiffused pattern (Fig. 1E). Taken together, these data demon-strate that HBx-NES is functional in Crm1-mediated nuclearprotein export.

Interaction between Crm1 and HBx—Nuclear export recep-tor Crm1 is known to interact with proteins containing NESsand to mediate nuclear protein export. The majority of Crm1protein is found in the nucleus of normal cells (39). Consis-tently, every NHF cell displays an endogenous nuclear-diffusedCrm1 distribution (Fig. 2). When HBx was expressed tran-siently in NHF, Crm1 was often found in the cytoplasm with apunctate staining pattern. Approximately 39% 6 2 of the HBx-expressing NHF cells displayed co-localization of Crm1, and

over 99% of cytoplasmic punctate Crm1 signals were co-local-ized with HBx (Fig. 2A). The punctate staining patterns con-taining both Crm1 and HBx signals were not always the samein appearance (Fig. 2A, panels c-l). These results suggest thatthe cytoplasmic HBx and Crm1 are not necessarily associatedwith a single subcellular component. In contrast, no cytoplas-mic co-localization was found in NHF cells expressing HBx-NESM. These data indicate that HBx was able to partiallyretain Crm1 in the cytoplasm and suggest that HBx binds toCrm1 in vivo. Consistently, in vitro translated HBx can phys-ically interact with in vitro translated Crm1 (Fig. 2B).

HBx-mediated Activation of NFkB Is Dependent on the Pres-ence of NES—HBx can activate NFkB and other cellular tran-scription factors including NF-AT and MAP kinase cascade,although the mechanism of activation is unknown (14, 40, 41).Because NFkB is inactivated normally through its binding toIkBa which is sequestrated in the cytoplasm by Crm1-medi-ated nuclear export (42), we examined whether HBx-NES isresponsible for the activation of NFkB. Over 97% of normaldividing NHF cells display a predominant localization of NFkBin the cytoplasm (Fig. 3A). In contrast, ;55 6 7% of HBx-expressing NHF cells show a preferentially nuclear distribu-tion of NFkB, while an NES mutated HBx (HBxNESM) has lostthis activity (Fig. 3, A and B). Next, we used a luciferasereporter that contains NFkB responsive elements (pNFkB-Luc)to investigate if the HBx-induced nuclear localization of NFkBcorrelates with its activity. pNFkB-Luc was co-transfected with

FIG. 2. Interaction between the nuclear export receptor Crm1 and HBx. A, co-localization of HBx and Crm1 in the cytoplasm. Normalhuman primary fibroblasts were immunostained with anti-Crm1 and detected by an fluorescein isothiocyanate-conjugated secondary antibody (a,c, g, i, and k). Cells without (a and b) and with transient expression of HBx (c-l) were immunostained with anti-HBx and detected by a TexasRed-conjugated secondary antibody (d, h, j, and l). The following images are from the same filed to visualize the co-localization of HBx and Crm1:panels c-f; panels e and f; panels g and h; panels i and j; panels k and l. B, HBx is able to bind to Crm1 in vitro. In vitro translated hCrm1 wasincubated without (lane 2) or with in vitro translated HA-tagged HBx (lane 3). Following immunoprecipitation with anti-HA antibody, Crm1 wasdetected by Western blot analysis with anti-Crm1 polyclonal antibody. One-tenth of the in vitro translated hCrm1 protein was loaded in parallelas input (lane 1).

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HBx or HBxNESM into an hTERT-immortalized NHF (NHF-hTERT) or a hepatocellular carcinoma cell line (Hep3B) in theabsence or presence of TNFa, an agent known to sensitizeHBx-mediated transactivation (9). Consistently, HBx alone in-duced the luciferase activity weakly which could be furtherenhanced by the treatment of TNFa in both NHF-hTERT andHep3B cells. In contrast, HBxNESM is devoid of its ability toincrease luciferase activity even in the presence of TNFa (Fig. 3C).

Cytoplasmic Localization of Crm1 Associated with HBV-in-fected Liver Samples—To investigate if Crm1 is sequestrated

cytoplasmically in liver samples from chronic active hepatitispatients with HBV infection, we performed immunohistochem-istry analysis of Crm1 in frozen tissue sections or in paraffin-embedded sections by anti-Crm1 polyclonal antibody. A total of12 liver samples with samples from 5 normal individuals wereincluded in this study. The presence of HBV DNA includingCore, preS, and HBx genes in these samples was verified byPCR (data not shown). Among 7 HBV positive cases, liversamples show chronic active hepatitis with grades 1 to 2 activ-ity (Table I). While 5/5 normal samples revealed nuclear Crm1staining exclusively, 6/7 HBV samples showed cytoplasmicCrm1 staining (Fig. 4 and Table I) in hepatocytes. An anti-NFkB antibody was also used to determine the status of NFkBin these liver samples. Again, normal liver samples revealedmainly a cytoplasmic localization of NFkB while HBV positiveliver samples showed a nuclear distribution of NFkB. Theabove findings provide an in vivo physiological significance forthe presence of a cytoplasmic sequestration of Crm1 accompa-nied by an activation of NFkB associated with HBV infection.

DISCUSSION

We have demonstrated that the oncogenic HBx contains afunctional NES, and that this NES is required for the HBx-mediated activation of the NFkB signaling cascade. We alsohave shown that HBx is able to bind to and sequester Crm1 inthe cytoplasm, which correlates with the nuclear localizationand activation of NFkB. Furthermore, liver samples from HBV-infected patients with chronic active hepatitis, a condition pre-disposing individuals for the development of liver cancer, alsodisplay cytoplasmic sequestration of Crm1 and nuclear local-ization of NFkB. It is possible that HBx may use a Crm1-de-pendent mechanism to modulate cellular gene transcription.Our data provide a plausible model whereby HBx may induceoncogenicity through the modulation of the Crm1-mediatedpathway.

The studies described in this report may offer a commonmechanism to explain the pleiotropic effects of HBx. First,Crm1 transports and controls many cellular proteins includingNFkB, NF-AT, MAPKK, and p53, whose activities are known tobe regulated by HBx (14, 17, 40, 41, 43). Second, Crm1 alsoregulates cytoplasmic mRNA transport. Inactivation of Crm1by HBx, in principle, would alter the stability of mRNA therebyinfluencing gene expression. Consistent with this hypothesis isour findings that HBx is able to sequester Crm1 in the cyto-plasm. The HBx-NES is essential for sequestering Crm1 in thecytoplasm and for the activation of NFkB. Inactivation ofCrm1-mediated nuclear protein export is known to activatethis target (44). Therefore, the promiscuous transactivation-mediated by HBx may be a secondary effect resulting fromalteration of Crm1 function.

An alternative mechanism for HBx to activate NFkB isthrough the degradation of IkBa in the cytoplasm by inducingits phosphorylation and ubiquitination. However, we did notobserve any detectable degradation of IkBa in cells expressingHBx by Western blot analysis with anti-IkBa antibody (datanot shown). This also correlates with our results that HBxalone only displays a weak activation of NFkB that can beenhanced significantly by TNFa (Fig. 3C). It is possible that asmall degree of dissociation of the NFkB-IkBa complex, followedby the degradation of IkBa induced by HBx in the cytoplasm,may contribute to the nuclear localization of NFkB. A recentstudy by Weil and colleagues (45) indicate that HBx may inducethe nuclear import of NFkB/IkBa by binding to IkBa. Interest-ingly, the region of IkBa that is critical for HBx-induced nuclearimport of IkBa contains a functional NES (45). These findings areconsistent with our data that HBx-induced nuclear localization ofNFkB/IkBa is dependent on the IkBa-NES.

FIG. 3. HBx-mediated activation of NFkB is dependent on afunctional nuclear export motif. A, normal primary human fibro-blasts were expressed transiently without (a-c), or with HBx (d-f) orHBxNESM (g-i) for 24 h. Cells were fixed and stained with anti-NFkB.Texas Red-conjugated secondary antibody was used to visualize NFkB(a, d, and g). The HBx signal was detected directly with an fluoresceinisothiocyanate filter (e and h). Percentages of cells with nuclear stainingof NFkB that are positive for HBx are shown B. C, a luciferase reportercontaining 4 NFkB responsive elements was transfected with HBx or anHBx mutant (HBxNESM, L98A, G99A, L100A) into a telomerase-im-mortalized human fibroblast line (NHF-hTERT) or a hepatocellularcarcinoma cell line (Hep3B). Cells were then treated with or without 3ng/ml TNFa for 24 h. The luciferase activity was measured and datawere normalized by an internal Renilla luciferase vector.

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HBx is thought to be small enough to diffuse passivelythrough the nuclear pore complex. The fact that this oncogenicviral protein has acquired NES activity, is preferentially local-ized in the cytoplasm, and modified nuclear export implies thatnuclear export may be an important target for viral-mediatedoncogenesis. Interestingly, cellular oncoproteins such as c-Abland Mdm2 are known to be involved in a Crm1-dependentnuclear export. Analogous to viral hepatitis oncoprotein, thecellular oncoproteins, in principle, may induce neoplastictransformation also through the disruption of the Crm1-medi-ated mechanism. It is known that TPR and CAN are nuclearpore complex-associated proteins that act as the docking sitesand are essential for Crm1-mediated nuclear export (27). Ear-

lier findings demonstrated that the TPR and CAN genes aremutated through translocation in thyroid carcinoma, gastriccarcinoma, and acute myeloid leukemia (46–52). We also foundthat cytoplasmic sequestration of Crm1 is associated fre-quently with HCC (data not shown). In addition, Crm1 and Ranmay play a role in mitosis initiation (29–31). It is possible thatthe inactivation of Crm1 and Ran may induce genomic insta-bility, a predisposing factor for cancer development. Thus, wepostulate that the inactivation of the Crm1-mediated nuclearexport is a common event during viral and cellular oncogenesis.

Acknowledgments—We thank Dr. Gerard Grosveld for generous giftsof anti-Crm1 antibody and the Crm1 cDNA, Dr. Barbara Wolff for theleptomycin B, Dr. John Brady for the pNFkB-Luc plasmid, Dr. JudyCampisi for the NHF-hTERT cell line, and Dr. Rodney Markin for thehuman liver samples. We are grateful to Dr. Curtis Harris and mem-bers of his group for scientific support, Susan Garfield for assistance onthe confocal imaging analysis, and Dorothea Dudek for excellent edito-rial assistance.

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TABLE ICytoplasmic sequestration of Crm1 and nuclear localization of NFkB associated with HBV-infected liver samples

Casenumber Histology Gradea Virus

StatusbCrm1 NFkB

Localizationc Score Localization Score

1 Normal 0 Negative Nuclear 41 Cytoplasmic 312 Normal 0 Negative Nuclear 41 Cytoplasmic 213 Normal 0 Negative Nuclear 41 Cytoplasmic 114 Normal 0 Negative Nuclear 41 Cytoplasmic 315 Normal 0 Negative Nuclear 41 Cytoplasmic 216 CAH 2 HBV Cytoplasmic 41 Cytoplasmic/nuclear 217 CAH 2 HBV Nuclear/cytoplasmic 41 Nuclear 218 CAH 2 HBV Cytoplasmic 41 Nuclear 119 CAH 2 HBV Cytoplasmic 41 Nuclear 1110 CAH 2 HBV Cytoplasmic 41 Nuclear 2111 CAH 1 HBV Cytoplasmic 41 Nuclear 2112 CAH 1 HBV Cytoplasmic 41 Nuclear/cytoplasmic 21

a Histology was determined according to the criteria of Scheuer. CAH, chronic active hepatitis.b The virus status was determined by serological tests and by determining the presence of preS, HBx, and core genes in these tissues by PCR.

HBV, hepatitis B virus.c Frozen sections were subjected to immunohistochemical analysis by the anti-Crm1 or anti-NFkB antibodies. Anti-Crm1 or anti-NFkB reactivity

was evaluated when crisp brown nuclear or cytoplasmic staining was detected, and scored as the following: a score of 0, no staining; 11, when ,10%of cells were stained; 21, .10 to ,25% of cells were stained; 31, .26 to ,50%; and 41, .50%. A score of (11) staining or higher was consideredreactive. The following criteria were used in determining protein distribution: nuclear, nuclear distribution only; cytoplasmic, cytoplasmicdistribution only; nuclear/cytoplasmic, predominant nuclear with some cytoplasmic distribution; cytoplasmic/nuclear, predominant cytoplasmicwith some nuclear distribution.

FIG. 4. Cytoplasmic sequestration of Crm1 and nuclear localiza-tion of NFkB associated with HBV-infected liver samples. Frozensections of a normal (A, C, and E) and an HBV positive (B, D, and F) liversamples were analyzed by H & E staining (A and B), anti-Crm1 antibodystaining (C and D), or anti-NFkB antibody staining (E and F). Represent-ative images are shown. Arrows indicate representative cells that arepositively stained by anti-Crm1 or anti-NFkB antibodies. Primary mag-nifications: H & E images, 3200; IHC images, 3400.

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Minoru Yoshida and Xin Wei WangMarshonna Forgues, Aizen J. Marrogi, Elisa A. Spillare, Chuan-Ging Wu, Qin Yang,

Export PathwayInteraction of the Hepatitis B Virus X Protein with the Crm1-dependent Nuclear

doi: 10.1074/jbc.M101259200 originally published online April 3, 20012001, 276:22797-22803.J. Biol. Chem. 

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