HBV X Protein Targets Hbubr1, Which Induces Dysregulation of the Mitotic Checkpoint

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Oncogene (2008) 27, 3457–3464 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint

S Kim1, S-Y Park1, H Yong1, JK Famulski2, S Chae1, J-H Lee1, C-M Kang3, H Saya4,GKChan2 andH Cho 1

1Department of Biochemistry, Ajou University School of Medicine, Suwon, Korea; 2Department of Oncology, University of Alberta, Edmonton, Alberta, Canada; 3Korea Institute for Radiological and Medical Sciences, Seoul, Korea and 4Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan

Accurate chromosomal segregation is monitoredby the addition, a defective mitotic checkpoint has also been mitotic checkpoint, andan increasedrate of chromosomal recognizedas a causative factor of CIN (Kops et al., missegregation leads to chromosomal instability (CIN). 2004). The mitotic checkpoint monitors microtubule Here, we demonstrate that the HBV X protein (HBx) attachment at kinetochores during mitosis and prevents binds BubR1, a component of the mitotic checkpoint cells with unalignedchromosomes from proceeding complex andco-localizes with BubR1 at the kineto- to anaphase by inhibiting the anaphase-promoting chores. HBx binding to BubR1 attenuates the association complex/cyclosome (APC/C) (Fang et al., 1998; Fang, between BubR1 andCDC20, an activator of the 2002). In mammalian cells, APC/C is regulatedby the anaphase-promoting complex/cyclosome (APC/C) and mitotic checkpoint complex (MCC), which is composed induces slippage of mitotic arrest in the presence of of BubR1, Bub3, Mad2 and CDC20 (Sudakin et al., microtubule poisons. In addition, HBx binding to BubR1 2001). Mouse embryonic fibroblasts (MEFs) derived results in the accumulation of lagging chromosomes from heterozygous BubR1 þ /À, Mad2 þ /À or CENP-E þ /À andchromosome bridges.In contrast, a C-terminally mice exhibit chromosome missegregation andaneu- truncatedHBx mutant (HBx 1–100) fails to bindBubR1 ploidy (Michel et al., 2001; Dai et al., 2004; Weaver anddoesnot cause aberrant chromosomal segregation. et al., 2007). This provides a novel mechanism for dysregulation of Hepatocellular carcinoma (HCC) is a leading cause of the mitotic checkpoint by a viral pathogen linking it to death worldwide. One of the primary etiological factors the accumulation of chromosomal instability in HBV- for the development of liver cancer is chronic infection associatedhepatocarcinogenesis. with hepatitis B virus (HBV) andCIN plays an Oncogene (2008) 27, 3457–3464; doi:10.1038/sj.onc.1210998; important role in liver carcinogenesis. Analysis of publishedonline 14 January 2008 primary liver tumors identified two distinct groups basedon CIN status, with one group showing high Keywords: HBV X; BubR1; chromosome instability; frequencies of allelic imbalance andchromosomal mitotic checkpoint loss being closely associatedwith HBV infection (Laurent-Puig et al., 2001). This is likely associated, at least in part, with the observation that integration of the HBV genome causes rearrangement of cellular DNA Introduction (Henderson et al., 1988). In addition, our group and others have observedthat the HBV X protein (HBx) Chromosome instability (CIN) is directly related to viral oncoprotein is directly involved in hyperamplifi- aneuploidy, which is regarded as one of the hallmarks of cation of centrosomes, which leads to chromosome cancer (Sen, 2000). CIN can be causedby an increased aberrations (Forgues et al., 2003; Yun et al., 2004; rate of chromosomal missegregation during mitosis Fujii et al., 2006). (Gollin, 2004), andabnormal numbers of centrosomes HBx is a multifunctional protein (Murakami, are associatedwith multipolar spindles that cause 2001), which deregulates cell cycle checkpoint controls chromosomal missegregation (Lingle et al., 2002). In (Madden and Slagle, 2001) potentially including the G2/M checkpoint (Lee et al., 2002). Notably, HBx-expressing cells become multinucleatedunder Corresponding authors: Professor Dr H Cho, Department of Biochemistry, Ajou University School of Medicine, 5 Wonchon-dong, microtubule-damaging conditions (Lee et al., 2002), Yeongtong-gu, Suwon, Kyunggi-do, 443-721, Republic of Korea. raising the possibility of mitotic checkpoint defects. E-mail: [email protected] and Here, we demonstrate that (1) HBx interacts with Professor Dr GK Chan, Department of Oncology, University of the BubR1 mitotic checkpoint protein andinterferes Alberta, Cross Cancer Institute, Edmonton, Alberta, Canada. E-mail: [email protected] with the binding of BubR1 to CDC20 and (2) HBx Received10 August 2007; revised9 November 2007; accepted20 binding to BubR1 triggers aberrant chromosomal November 2007; publishedonline 14 January 2008 segregation. HBV X targets hBubR1 SKimet al 3458 Results However, neither the LexA-fused Cdc20, Mad2 nor Bub3 interactedwith B42-HBx (Supplementary HBx targets hBubR1 Figure 1), confirming the specific interaction of Some viral oncoproteins such as Tax andsimian virus40 HBx with BubR1. We further foundthat yeast (SV40) T antigen interact with mitotic checkpoint expressing an hBubR1 C-terminal truncation construct components andthese interactions contribute to aber- (LexA-hBubR11À467) andB42-HBx failedto grow on the rant mitotic phenotypes (Cotsiki et al., 2004; Liu et al., selection media, suggesting that the N-terminal region 2005). Similarly, HBx-induced multinucleated cells of hBubR1 is not involvedin HBx binding.Binding under microtubule-damaging conditions (Lee et al., between a LexA-fusedC-terminal fragment of 2002) couldbe potentially mediatedthroughdefects in CENP-E1958À2662 andits known bindingpartner, the mitotic checkpoint. To examine this possibility, we B42-BubR1408À1050, was usedas a positive control first useda targetedyeast two-hybridassay to detect (Chan et al., 1998; Figure 1a). binding between HBx and any of the MCC components. We confirmedthe bindingof HBx andBubR1 in A full-length cDNA encoding HBx1À154 was clonedinto mammalian cells. Since mammalian MCC components a D-galactose (gal)-inducible B42 activation domain were originally identified in HeLa cells (Sudakin et al., vector (pJG4-5). We foundthat a fusion protein 2001), we transfectedHeLa cells with a pMyc-tagged combining LexA andfull-length hBubR1 1À1050 interacted HBx construct. Forty-eight hours post transfection, cell with B42-HBx to yieldcolonies in leucine-free media lysates were immunoprecipitatedwith either an anti- andwas positive on an X-Gal plate (Figure 1a). mouse IgG antibody or a mouse anti-Myc antibody, followedby western blotting using an anti-BubR1 antibody. Endogenous hBubR1 was present in immune a LexA-∆BubR1 LexA-BubR1 complexes obtainedwith the anti-Myc antibody, which B42-HBx B42-HBx targetedthe Myc-taggedHBx, but not with anti-mouse LexA-∆BubR1 IgG (Figure 1b). A reciprocal immunoprecipitation B42-HBx experiment using an anti-BubR1 antibody also con- LexA-BubR1 LexA-CENP-E LexA-CENP-E firmedthe interaction between HBx andhBubR1 B42-HBx B42-BubR1 B42-BubR1 (Figure 1b). To examine whether these interactions occur in a cell-cycle-dependent manner we next prepared cell lysates from both asynchronously growing cells (I: mainly interphase) andmitotic cells (M: obtained 1 c bR after nocodazole treatment for 12 h). Immunoprecipi- gG My u α-I α- α- B tation with the anti-BubR1 antibody revealed that HBx : : : IP IP IP b boundhBubR1 to similar degreesin interphase and ++ +pMyc-HBx mitotic phase cells (Figure 1c). Since BubR1 functions as 130 α BubR1 a mitotic checkpoint protein, we analysedstrictly mitotic cell lysates in the following experiments. These 20.1 α − Myc results are the first indication that BubR1 can be targetedby the HBx viral oncoprotein.

I M

-+-+pMyc-HBx HBx co-localizes with BubR1 at the kinetochores c Activation of the mitotic checkpoint may require the α − 20.1 Myc proper assembly of mitotic checkpoint components at the kinetochores (Cleveland et al., 2003). Since HBx 130 α − BubR1 binds BubR1, we hypothesized that HBx might localize to the kinetochores during mitosis. To test this notion, α − IP: BubR1 we utilizedHBx-expressing ChangX cells (Yun et al., Figure 1 HBV X protein (HBx) interacts with BubR1. (a)Ina 2004) previously developed in our lab and SNU368 cells yeast two-hybridassay, yeast expressing LexA-BubR1 1À1050 and that contain the HBV genome andexpress HBx B42- HBx1À154 resultedin growth in medialacking leucine (left) andblue colonies on the X-gal plate (right). Interaction between protein(Kim et al., 2007). Specific expression of HBx LexA-CENP-E andB42-BubR1 was usedas a positive control. protein in ChangX andSNU368 cells but not in Huh7 (b) The pMyc-HBx plasmidwas transfectedinto HeLa cells. Cell andChang cells were shown (Figure 2c). We first lysates were immunoprecipitated(IP) with either goat anti- confirmedthat BubR1 in SNU368 cells co-localizedat mouse-IgG, anti-Myc or anti-BubR1 antibody. The precipitates the kinetochores of the condensed chromosomes with were analysedby western blotting (WB) with anti-BubR1 antibody (top) andwith anti-Myc antibody(bottom). ( c) The pMyc-HBx the kinetochore-specific marker, CREST (Moroi et al., plasmidwas transfectedinto HeLa cells. Interphase cell lysates 1980; data not shown). Notably, a portion of the HBx were preparedat 48 h post transfection (I; interphase). Mitotic cell protein co-localizedwith hBubR1 at the kinetochores in lysates were preparedafter 12 h of nocodazole (100 ng ml À1) SNU368 cells as well as in ChangX cells (Figure 2a). challenge at 36 h post transfection (M, mitosis). The immunoprecipitates obtainedwith anti-BubR1 antibodywere analysedby Magnification of the kinetochore staining (Figure 2a, western blotting with anti-Myc antibodies (top) and anti-BubR1 inset) clearly indicated co-localization of HBx and antibodies (bottom). hBubR1 at the kinetochores. We next examinedwhether

Oncogene HBV X targets hBubR1 SKimet al 3459

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X 8 g g 7 36 an an h U h N Ch C Hu S - 100 200 (pmol)

20.1 HBx 130 BubR1 66 45 α-tubulin β-Actin

Figure 2 HBV X protein (HBx) co-localizes with BubR1 at the kinetochores. (a) Asynchronously growing ChangX andSNU368 cells were fixedwith a mixture of methanol/acetone andimmunofluorescence staining was carriedout using rabbit anti-HBx (FITC) and mouse anti-BubR1 (Cy3) antibodies with DAPI staining (blue). Fluorescence images of mitotic cells were captured by confocal microscopy. The inset image depicts the magnified region of interest. Arrows in the inset indicate kinetochore co-localization. Scale bar ¼ 10 mm. (b) ChangX cells were transfectedwith siRNA duplexes targeting the hBubR1 sequence using Lipofectamine 2000 and immunofluorescence staining was carried out. Arrows indicate the spindle poles. (c) HBx expression was determined by western blotting. (d) Expression levels of BubR1 were determined 48 h after transfection of siRNA duplex targeting hBubR1. Molecular weight markers (kDa) are indicated at the left side of the panels of western blot analysis.

BubR1 is requiredto tether HBx at kinetochores by BubR1 (data not shown). Thus, the interaction between knockdown of BubR1 expression. The hBubR1 siRNA HBx andBubR1 may contribute to the kinetochore duplex signiﬁcantly reduced BubR1 expression in localization of HBx. ChangX cells (Figure 2d) and we found that the kinetochore localization of HBx was diminished in cells lacking kinetochore hBubR1 (Figure 2b, lower panel). HBx reduces the association between hBubR1 and In contrast, centrosomal localization of HBx (Fujii CDC20 et al., 2006) appearednot to be affectedby BubR1 We next examinedthe HBx-bindingregion of BubR1. siRNA (Figure 2b, arrows). Localization of CDC20 to N-terminally truncatedhBubR1 408À1050 (BubR1DN, har- kinetochores appearedto be stable in cells depletedof boring the CDC20 binding site and the kinase domain)

Oncogene HBV X targets hBubR1 SKimet al 3460 andC-terminally truncatedhBubR1 1À685 (BubR1DC, decrease in HBx binding, whereas pGFP-BubR1DC lacking the kinase domain) were generated as fusion retainednearly all of its HBx-bindingactivity (Figure 3b). proteins with GFP (Figure 3a; Chan et al., 1999) and Both deletion mutants shared amino acids 408–685 of co-transfectedwith the pMyc-HBx vector. Cell lysates hBubR1; this region contains the CDC20 binding site were immunoprecipitatedwith an anti-GFP antibody, (Fang, 2002), suggesting that HBx andCDC20 share a andthe presence of HBx in the immunocomplexes was common binding region within hBubR1 (Figure 3a). determined. Both BubR1 deletion mutants retained the HBx mainly consists of two domains, a regulatory ability to bindHBx; pGFP-BubR1 DN showedsome domain (1–50) and a transactivation domain (51–154;

1 410 464 525 700 731 1050 a. a N CDC20 binding Kinase domain C

Bub3-binding putative HBx binding 467 685 BubR11-1050 BubR1408-1050(∆N) BubR11-685(∆C) BubR11-467

N C ∆ ∆ 1 1 1 R R R 4 0 4 b b b 5 0 0 5 1 5 1 -1 u u u - - - 1 B 1 1 1 5 -B - -B P P P P ly x x x x F F F n B B B B G G G GF o H H E E H H p E E c c c c c p p p y y y y y M M M M ++ + + pMyc-HBx p p p pM p Input 24.1

97 14.3 α−Myc 66 Input α−GFP 45 bound HBx IP : α−BubR1 30 WB : α− Myc bound ∆HBx Input α−Myc

IP : α−GFP bound HBx WB: α−Myc

-+pMyc-HBx -+pMyc-HBx 130 IP : α−CDC20 Input 130 α−BubR1 WB: α−BubR1 66 20.1 α−Myc α−CDC20 66 IP : α− α−BubR1 CDC20 45 α−Bub3

130 α−BubR1

Figure 3 HBV X protein (HBx) inhibits the binding between BubR1 and CDC20. (a) A schematic representation of hBubR1, its constructs andits functional domains. ( b) The full-length anddeletionconstructs of pEGFP-BubR1 (BubR1 DN; hBubR1408À1050, BubR1DC; hBubR11À685), pEGFP (vector control) andpMyc-HBx were co-transfectedinto HeLa cells andexpression levels were shown (input). Cell lysates were immunoprecipitatedwith anti-GFP antibodyandthe presence of HBx in the resulting pellets was determined (bottom). (c) Full-length anddeletionHBx constructs were transfectedinto HeLa cells andanalysedfor expression (input). Cell lysates were immunoprecipitated with anti-BubR1 antibody and the presence of HBx in the pellets was determined (bottom). (d) HeLa cells were transfected for 36 h and followed by nocodazole treatment for 12 h. The cell lysates were divided and immunoprecipitated with either anti-BubR1 antibody (left) or anti-CDC20 antibody (right). The immunoprecipitates were resolved and analysed by immunoblotting with the indicated antibodies. Molecular weight markers are indicated in each panel. Arrowheadindicates the heavy chain of immunoglobulin G (IgG).

Oncogene HBV X targets hBubR1 SKimet al 3461 Lin et al., 1997). We foundthat the HBx 1À50 and transiently transfectedwith HBx (Figure 4c). Thus, HBx1À100 fragments didnotbindto hBubR1 (Figure 3c). these results demonstrated that HBx expression induced In contrast, a stable interaction between hBubR1 and mitotic slippage in cells under microtubule damaging HBx1À154 was detected, and the HBx51À154 fragment was conditions. foundto retain comparable BubR1 bindingability relative to the amount of expressedprotein (Figure 3c). Binding ofHBx to hBubR1 increases the rate of Since HBx andCDC20 seem to share a bindingregion chromosome instability within hBubR1, we investigatedwhether HBx competes Since perturbation of the mitotic checkpoint in cells can with CDC20 for binding to hBubR1. HeLa cells are increase the rate of chromosomal missegregation during usedbecause it has a robust mitotic checkpoint. First, mitotic progression (Cleveland et al., 2003), we evalu- we observedthat BubR1 efficiently recruits CDC20 in atedthe frequency of aberrant chromosomal segregation HeLa cells treatedwith nocodazole(Supplementary in HBx-expressing cells by aceto-orcein staining. In Figure 2). In the BubR1 immunoprecipitates, we found these experiments, we have only included the aberrant that the interaction between BubR1 andCDC20 was chromosomal segregation derived from bipolar mitotic significantly reduced when HBx was present (Figure 3d). spindle poles and excluded those driven from multiple Densitometric analysis revealedthat BubR1 bindingto centrosomes (Forgues et al., 2003; Yun et al., 2004). The CDC20 in the presence of HBx was reduced to 27% of number of cells with unalignedchromosomes at the control value. Three other independent experiments metaphase (Figure 5a, arrows) andchromosome bridges consistently showed reduced binding (27–52% of at anaphase/telophase (Figure 5b, arrows) were sig- the control value) between BubR1 andCDC20 in the nificantly increasedin cells expressing HBx 1À154 and presence of HBx. Reciprocal immunoprecipitations HBx51À154. Less than 5% of control metaphase cells had using an anti-CDC20 antibody also showed that the unalignedchromosomes, while the percentage seen CDC20–BubR1 interaction was reduced in the presence in metaphase HBx1À154- or HBx51À154-expressing cells of HBx (Figure 3d, right panel). However, HBx did not was elevatedat least threefold(* P 0.05, **P 0.005, affect the interaction between BubR1 andBub3 o o Student’s t-test). Similarly, mitotic bridges were seen in (Figure 3d, left panel). Together, these results demon- B10% of control cells, whereas those rates were strate that HBx interferes with BubR1 binding to significantly higher in cells transfectedwith HBx 1À154 CDC20 in the MCC of mammalian cells, suggesting andHBx 51À154 (Figure 5b). In contrast, expression of that the binding of HBx to BubR1 may disturb mitotic hBubR1 binding-deficient HBx1À50 or HBx1À100 did not checkpoint function. alter the occurrence of aberrant chromosome segrega- tions. Taken together, these results indicate that binding of HBx to BubR1 significantly increases aberrant Compromised mitotic checkpoint in cells expressing chromosome segregation. HBx protein A compromisedmitotic checkpoint ledto mitotic slippage in BubR1 þ /À MEFs expressing low BubR1 level (Dai et al., 2004). Similarly, the reduced binding of Discussion hBubR1 to CDC20 in the presence of HBx might also induce defective mitotic checkpoint function. In fact, we We have demonstrated for the first time that BubR1, a foundthat HBx-expressing ChangX cells escapedfrom key component of the MCC, is a target of the HBx viral mitotic arrest after nocodazole treatment (Figure 4a). At oncoprotein. Importantly, the HBx–BubR1 interaction 18 h, B5% of ChangX cells escapedfrom mitotic arrest was foundto disruptCDC20 bindingin the MCC and (Supplementary Figures 3Ab andB), whereas a sig- HBx-expressing cells underwent faulty mitotic progres- nificant portion of them (25–30%) also underwent cell sion. Thus, the interaction between HBx andBubR1 death (Supplementary Figures 3Ac, Ad and B) as provides a novel mechanism that may account, at least determined by aceto-orcein staining. At 24 h, 75% of in part, for increasedchromosomal instability during Chang cells remainedarrestedat mitosis whereas the HBV-mediated carcinogenesis. mitotic index in ChangX cells was reduced to 20% even Our findings add to the list of viral proteins that are in the presence of nocodazole (Supplementary Figure known to interact with mitotic checkpoint proteins. 3B). Mitotic slippage during metaphase to anaphase SV40T antigen interaction with Bub1 promotedendor- transition can be further characterizedby the APC/C- eduplication under nocodazole treatment (Cotsiki et al., dependent cyclin B and securin degradation. Since 2004). Tax was reportedto bindMad1(Jin et al., 1998) prolongedtreatment of nocodazolesensitizedChangX andrecently to the CDC20-associatedAPC/C (Liu cells to death, we next synchronized cells by the double et al., 2005), prematurely activating the APC/C. How- thymidine block (DTB) method and added nocodazole ever, the mechanisms of action through which these viral to cells at G2/M phase to shorten the exposure to oncoproteins affect the mitotic checkpoint andtheir nocodazole. Immunoblot revealed that levels of cyclin B functional consequences are poorly characterized. andsecurin in Chang cells were maintaineduntil 10 h We have shown that binding of HBx to BubR1 whereas those in ChangX cells were already reduced disrupts mitotic checkpoint function. The HBx interac- after 4–6 h under nocodazole challenge (Figure 4b). We tion domain of BubR1 (amino acids 408–685) overlaps also confirmed early degradation of securin in cells with the CDC20 binding domain of BubR1 (Figure 3a

Oncogene HBV X targets hBubR1 SKimet al 3462

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02 46810(h) 024 6 810(h)

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Relative cyclinB level 0.2 0.2 Relative securin level 0 0 0246810(h) 0246 810(h) Time after nocodazole challenge Time after nocodazole challenge

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Figure 4 Compromisedmitotic checkpoint in cells expressing HBV X protein (HBx). ( a) A representative phase-contrast image of Chang andChangX cells 14 h after nocodazoletreatment. ( b) ChangX cells were synchronized by the double thymidine block (DTB) method and nocodazole was added 10 h after DTB release. Expression proﬁles of cyclin B and securin in Chang and ChangX cells are shown andanalysedby densitometry. ( c) Chang cells transfectedwith pMyc-HBx were treatedwith nocodazole.Expression proﬁles of securin, HBx and b-actin were analysedby immunoblotting.

andb; Fang, 2002). Both Mad2andBubR1 have been thereby allowing mitotic slippage andan accumulation shown to directly bind CDC20 and inhibit the activity of of aberrant chromosomes. Thus, our ﬁndings provide the APC/C in vitro (Fang et al., 1998; Fang, 2002) with new insights into viral pathogen-induced mitotic BubR1 functioning as a much more potent inhibitor of dysregulation. CDC20-associatedAPC/C activity (Sudakin et al., 2001; Tang et al., 2001). Thus, the binding of HBx to BubR1 may interfere with the ability of BubR1 to bindCDC20 Materials andmethods andits inhibitory activity towardthe CDC20-associated APC/C. The potential importance of the HBx–BubR1 Plasmids and antibodies interaction is highlightedby the observation that cells The cDNA expression vectors encoding HBx mutants were expressing HBx cannot properly arrest in mitosis under subclonedinto pCMV-Myc. hBubR1 cDNA was subcloned microtubule damaging condition (Figure 4). BubR1 null into the Gateway pENTR vector (Invitrogen, Carlsbad, CA, mouse embryos failedto survive beyondday8.5 (Wang USA) andsubsequently transferredinto the pWSGFP-GW þ /À destination vector using LR clonase recombination reaction et al., 2004) but BubR1 MEF expressing 10–25% of (Chan et al, 1998). Rabbit anti-HBx antibody was made normal BubR1 levels showedenhancedmitotic slippage against a synthetic HBx peptide corresponding to residues (Baker et al., 2004; Dai et al., 2004). In our system, HBx 144–154 (Lab Frontier, Seoul, Korea). Antibodies for Myc expression may mimic the situation of low BubR1 level andCDC20 were purchasedfrom Santa Cruz Biotechnology in cells andlack the complete inhibition of CDC20, (Santa Cruz, CA, USA) andantibodiesfor BubR1 andBub3

Oncogene HBV X targets hBubR1 SKimet al 3463

18 60 16 * 50 14 ** ** 12 40 * 10 30 8 6 20 /anaphase cells /metaphase cells 4 10 2 % of chromosome bridges % of chromosome % of lagging chromosomes chromosomes % of lagging 0 0 p pM H H H H M HB H H H y B B B B y x B B B c x 1 x 1 x 1 x 5 c 1- x 1- x 1- x 5 -1 -5 -1 1 15 5 1 1- 54 0 00 -1 4 0 00 15 54 4

Figure 5 HBV X protein (HBx) results in the accumulation of chromosomal instability. At 48 h post transfection of different forms of pMyc-HBx, Chang cells were stainedwith aceto-orcein andthe numbers of unalignedchromosomes ( a) andmitotic anaphase bridges (b) were quantified. Three to six independent experiments were performed in which B100 mitotic cells were countedeach time. *Po0.05, **Po 0.005 by Student’s t-test. were from BD Transduction Laboratories (San Jose, CA, using protein G-Sepharose andanalysedby western blotting. USA). Antibodies for securin and cyclin B were from ZYMED The transfection efficiency using the pEGFP control construct (San Francisco, CA, USA) andSanta Cruz, respectively. was determined as 70–80% with both calcium phosphate methodandPEI method(datanot shown). Cell culture and synchronization HBx-transfectedChangX cells (Yun et al., 2004; Kim et al., Microscopy analysis 2007) andChang, HeLa andSNU 368 cells were maintainedin Dulbecco’s modified Eagle’s medium (DMEM) supplemented Cells were seeded onto coverslips, fixed with methanol:acetone (1:1) solution, andpermeabilizedwith PBS containing 0.075% with 10% fetal bovine serum (FBS; Life Technologies Co., TritonX-100 (Yun et al., 2004). Fixedcells were pre-incubated Grand Island, NY, USA) in a humidified CO2 incubator. SNU 368 cells were purchasedfrom the Korean Cell Line Bank in blocking solution, followedby incubation with primary 1 (Seoul, Korea). antibodies at 4 C andprobedwith fluorescein-conjugated antibody. Images were captured under confocal microscopy (LSM510, Zeiss, Jena, Germany). For the analysis of Yeast two-hybrid assay chromosome aberration, the nuclei were stainedwith 2% Yeast strains as well as yeast expression vectors pEG202, aceto-orcein solution 48 h post transfection with different HBx pJG4-5 andpSH18-34 were providedbyDr E Golemis (Fox deletion mutants and numbers of mitotic cells with aberrant Chase Cancer Center, PA, USA). The hBubR1, hBub3, chromosomes were determined. hMad2 and CDC20 cDNAs were fused into pEG202 containing the LexA DNA binding domain, and HBx cDNA was fusedinto pJG4-5 harboring the D-galactose (gal)-inducible Acknowledgements B42 activation domain. The positive interaction was deter- minedby both b-galactosidase activity and leucine-dependent This work is supportedby grants of the National R&D growth. Program for Cancer Control, Ministry of Health andWelfare (0320010-2) andKorea Science andEngineering Foundation Transfection, immunoprecipitation and western blotting (R13-2003-019). GKC is supportedby the CanadianInstitute Cells were transfectedeither using the calcium phosphate of Health Research (CIHR) MOP 57723, the Alberta Cancer method(ProFection, Promega, Madison,WI, USA) or using Board, the Alberta Heritage Fund for Medical Research, the the polyethylenimine (PEI; Polysciences, Warrington, PA, Canadian Foundation for Innovation and the Alberta Science USA). For siRNA transfection, siRNA duplexes targeting the andResearch Authority. Salary of GKC is supportedby a hBubR1 sequence 50-AACAATACTCTTCAGCAGCAG-30 CIHR New Investigator Award. Stipend of JKF is supported were synthesizedandtranfectedinto ChangX cells using by a CIHR Canadian Graduate Scholarships and a Graduate lipofectamine 2000 (Invitrogen). Whole-cell lysates were Studentship from the Alberta Cancer Board. S Kim, S Park preparedby extracting cells with radioimmunoprecipitation andS Chae are supportedby the BK21 program, Korean (RIPA) buffer andincubatedwith various primary antibodies Ministry of Education. We thank Dr Dawn Macdonald for for 8–16 h at 4 1C andthe immune complexes were precipitated proofreading the manuscript.

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