Hepatitis B Virus Encoded X Protein Suppresses Apoptosis by

Aug 8, 2012 - ated inducer of death (AMID), also called PRG3 (p53- .... game, CA). ... The percentage of cell death was measured in EGFP-labeled cells...
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Hepatitis B Virus Encoded X Protein Suppresses Apoptosis by Inhibition of the Caspase-Independent Pathway Haiying Liu,‡,∥,#,∇ Yanzhi Yuan,§,∇ Hongyan Guo,∥,⊥ Keith Mitchelson,‡,∥,⊥ Ke Zhang,‡,∥ Lan Xie,‡,∥ Wenyan Qin,∥,⊥ Ying Lu,‡,∥ Jian Wang,§ Yong Guo,‡,∥ Yuxiang Zhou,*,†,‡,∥,⊥ and Fuchu He*,§ †

The State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China Medical Systems Biology Research Center, Department of Biomedical Engineering, Tsinghua University School of Medicine, Beijing 100084, China § The State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China ∥ National Engineering Research Center for Biochip Technology: Beijing, 18 Life Science Parkway, Beijing 102206, China ⊥ CapitalBio Corporation, 18 Life Science Parkway, Beijing 102206, China ‡

S Supporting Information *

ABSTRACT: Hepatitis B virus (HBV) encoded X protein (HBx) has been implicated in apoptotic and related pathogenic events during hepatocellular carcinoma. However, the underlying molecular mechanism through which HBx acts is largely unclear. We used tandem affinity purification under mild conditions to gain insight into the HBx interactome in HBV-producing HepG2.2.15 cells and identified 49 proteins by mass spectrometry that are potentially associated with HBx. Two of the key proteins of the caspase-independent apoptosis pathway were newly identified, apoptosis-inducing factor (AIF) and the homologous AMID (AIF-homologue mitochondrion-associated inducer of death). We confirmed the interactions of HBx with AIF and with AMID by reciprocal coimmunoprecipitation experiments, respectively. We observed the expression of HBx-reduced AIF-mediated apoptosis and HBx colocalization with AIF and AMID, principally in the cytoplasm. Furthermore, the elevated cytoplasmic levels of HBx could inhibit mitochondrion-to-nucleus translocation of AIF. Here, we present the first detailed molecular evidence that HBx can repress apoptosis via inhibition of the caspase-independent apoptosis pathway. This inhibition of apoptosis involves the repression of the mitochondrion-to-nucleus translocation of AIF, although tests with AMID were not conclusive. These findings provide important insights into the new mechanism of the apoptosis inhibition by HBV. KEYWORDS: hepatitis B virus, HBx, AIF, AMID, apoptosis, tandem affinity purification

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INTRODUCTION Hepatitis B virus (HBV) infection is a major risk factor contributing to the genesis of hepatocellular carcinoma (HCC). About 350 million people worldwide are chronically infected with HBV, and in high epidemic areas such as East Asia and sub-Saharan Africa, more than 80% of HCC patients are positive for hepatitis B surface antigen (HBsAg).1,2 The molecular mechanisms that link HBV infection to the development of HCC are not well understood. HBV encoded X protein (HBx) is widely reported to be involved in HBV pathogenesis and HCC development. HBx is a regulatory protein that plays important roles in viral transcription and replication. It also interferes with many normal cellular activities, such as cellular signal conduction, gene transcription, proliferation, and importantly with apoptosis.1 Several studies have demonstrated that HBx regulates apoptosis through a caspase-dependent pathway, intriguingly © 2012 American Chemical Society

either inhibiting or promoting apoptosis under different conditions.3−7 HBx can inhibit apoptosis by sequestering p53 in the cytoplasm, resulting in the activation of the PI3K pathway and up-regulation of the SAPK/JNK pathway, respectively.3−5 HBx is also reported to be pro-apoptotic by prolonging the stimulation of the N-myc and MEKK1 pathways, via interactions with c-FLIP and hyper-activation of caspase-8 and caspase-3, respectively.6,7 Recent studies have shown that HBx induces apoptosis or protects cells from apoptosis depending on the cellular status of NF-κB,5 and studies have associated some of the actions of HBx to the modulation of the mitochondrial permeability transition pore.8,9 In contrast to these numerous observations of HBxmodulated apoptosis involving the caspase-dependent pathway, Received: December 14, 2011 Published: August 8, 2012 4803

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EGFP, AMID-Myc, and AMID-EGFP expression vector plasmids.

to the best of our knowledge, there are no reports of HBx involvement in a caspase-independent apoptosis mechanism. The caspase-independent apoptosis pathway is an emerging mechanism through which cell apoptosis can occur. Susin et al. recently found that apoptosis-inducing factor (AIF) plays a key role in the caspase-independent pathway.10,11 AIF is a 67 kDa protein that is normally confined to mitochondria, yet upon apoptotic stimulation could be released by cleavage of calpains. A truncated 57 kDa form of AIF protein acts as the proapoptotic mediator, which translocates first from mitochondria into the cytosol and subsequently translocates from there into the nucleus, where it promotes chromatin condensation and DNA degradation.12 AIF-homologous mitochondrion-associated inducer of death (AMID), also called PRG3 (p53responsive gene 3), is a homologue of AIF that lacks the mitochondrial or nuclear localization sequence,13,14 although it has been reported to localize in mitochondria. Ectopic expression of AMID has been shown to induce apoptosis, via a caspase-independent mechanism similar to AIF.13 However, the molecular functions of AMID during caspase-independent apoptosis are still unclear. We have previously investigated HBV−host interactions at the nucleic acid level, demonstrating that cell microRNAs-372/ 373 are subverted to promote the expression of HBV through the targeting of nuclear factor I/B and that different interfering siRNAs can effectively inhibit HBV replication presumably with the involvement of different cellular genes.15,16 In this present study, we have examined the HBx protein-interactome in the HBV-producing hepatoma cell line, HepG2.2.15, which may model aspects of chronic HBV infection. As one major challenge for detailed understanding of the molecular mechanisms is the ability to identify low abundance HBx− protein complexes, we applied a tandem affinity purification (TAP)17,18 strategy, which involves the use of a high-affinity tag (TAP-tag) to provide for an enrichment of interacting molecules through the selective binding at two affinity domains: the protA domain (IgG-binding units of protein A) and the CBP domain (calmodulin-binding peptide), separated by a TEV (tobacco etch virus) protease recognition sequence. Subsequent MS-MS analysis of TAP-tag purified HBx complexes provided evidence of interactions between 49 cellular proteins and HBx, including novel interactions with two proteins, AIF and AMID. The binding of AIF with HBx was found to inhibit the translocation of AIF into the nucleus, resulting in an attenuation of apoptosis. This is the first report of the cytoplasmic interaction of HBx with both key members of the caspase-independent apoptosis pathway AIF and AMID and the inhibition of mitochondrion-to-nuclear translocation of AIF by HBx, causing an inhibition of apoptosis.

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Cell Culture and Transfection

HepG2 and HepG2.2.15 cells were maintained as described previously.15 These two cell lines were transfected with pZome1-C-HBx or pZome-1-C by the calcium phosphate method19 and maintained in 2 μg/mL Puro selective medium for 2 weeks to generate HBx-TAP-HepG2, TAP-HepG2, HBx-TAPHepG2.2.15, and TAP-HepG2.2.15 stable cell lines. The overexpression of HBx protein was confirmed by Western blotting with rabbit IgG, which bound to the Protein A of TAPtag. Transient transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. TAP-Affinity Purification

A modified TAP-affinity purification procedure based on the protocol of Rigaut and Puig was used, with approximate 6 × 108 HBx-TAP-HepG2.2.15 and TAP-HepG2.2.15 cells.17,18 Briefly, cells were incubated on ice for 30 min in 2 PCV (packed cell volume) of IPP150 (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, and 5% glycerol) supplemented with freshly added 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM DTT, and completely EDTA-free Protease Inhibitor (Roche, Basel, Switzerland). The lysates were then centrifuged at 10000g for 20 min at 4 °C. The supernatants were collected and incubated with IgG sepharose beads (Pharmacia, Uppsala, Sweden) for 2 h at 4 °C. Beads were washed three times with 5 mL of TEV cleavage buffer (IPP150 adjusted to 0.5 mM EDTA and 1 mM DTT) and then incubated with 100 units of TEV protease (Invitrogen) in 350 μL of TEV cleavage buffer overnight at 4 °C. The TEV cleavage products were recovered by gravity flow. Beads were then washed three times with 350 μL of IPP150 calmodulin binding buffer (IPP150 adjusted to 10 mM β-mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, and 2 mM CaCl2) per time, and the eluted wash buffer was collected and incorporated with corresponding TEV cleavage product. This solution was then incubated with calmodulin beads (Pharmacia) for 2 h at 4 °C. Beads were washed with 1 mL of IPP150 calmodulin binding buffer for three times. Proteins retained on the beads were eluted with 1.05 mL of IPP150 calmodulin elution buffer (IPP150 adjusted to 10 mM β-mercaptoethanol, 1 mM magnesium acetate, 1 mM imidazole, and 20 mM EGTA). The eluates were concentrated by trichloroacetic acid (TCA) precipitation and resuspended in 50 μL of IPP150. Protein Digestion

Precipitated proteins from HBx-TAP-HepG2.2.15 and TAPHepG2.2.15 cells were dissolved in SDS loading buffer, separated by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue R250 in parallel. The full lanes were cut into small bands, despite visible protein bands or not. For trypsin digestion, gel slices were cut into cubes, destained with 50% acetonitrile containing 25 mM NH4HCO3, and subjected to reduction and alkylation. Then, the gel plugs were lyophilized and immersed in 15 μL of 10 ng/μL trypsin solution in 25 mM NH4HCO3. The digestion was incubated at 37 °C for 15 h. Peptide mixtures were first extracted with 100 μL of 5% trifluoroacetic acid (TFA) at 40 °C for 1 h and then 2.5% trifluoroacetic acid/50% acetonitrile at 30 °C for 1 h. The extracted solutions were combined, lyophilized, and prepared for the following analysis.

MATERIALS AND METHODS

Plasmids

The HBx-encoding gene was constructed to the N-terminal of TAP-tag of pZome-1-C (Cellzome, Heidelberg, Germany), resulting in pZome-1-C-HBx. This gene was also cloned into plasmids pFLAG-CMV-2 (Sigma, St. Louis, MO) and pDsRed1-C1 (Clontech, Mountain View, CA), respectively, to produce HBx-Flag and HBx-DsRed expression vectors. The full-length AIF cDNA and AMID cDNA were cloned independently into pcDNA 3.1/Myc-His (Invitrogen, Carlsbad, CA) and pEGFP-N1 (Clontech) to produce the AIF-Myc, AIF4804

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Pittsburgh, PA) overnight at 4 °C. The immuno-complexes were washed three times with lysis buffer, released by boiling at 100 °C for 5 min in SDS sample buffer (65 mM Tris-HCl, pH 8.0, 10% glycerol, 2.3% SDS, 0.01% bromophenol blue, and 1% DTT), and analyzed by Western blotting.

LC-MS/MS Analysis

Lyophilized tryptic peptides were subjected to Nano-RPLCMS/MS using a LCQ Deca XP plus ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an NSI source. A Finnigan Surveyor HPLC (Thermo Finnigan) was employed with a nanoflow delivery system. The 10-port valve of mass spectrometer was equipped with two RP-C18 trap columns (300 μm i.d. × 5 mm, Dionex, Sunnyvale, CA), which allows for synchronous sample loading and desalting of one trap column, whereas the other is eluted to the MS through a analytical column (75 μm i.d. × 10 cm C18 PicoFrit with 15 μm ID pulled tip, New Objective, Woburn, United States). A stepwise mobile phase gradient of 0−40% B (A = 0.1% formic acid in water, and B = 0.1% formic acid in acetonitrile) for 60 min and 40−95% B for 15 min was employed with a flow rate of 300 nL/min. The LCQ-MS was operated under the following parameters: normalized collision energy of 35%, temperature of the ion transfer of 200 °C, and spray voltage of 1.8 kV. MS/MS acquisition for the three most intense ions following one full MS scan was set up with the following dynamic exclusion settings: repeat count of 2, repeat duration of 0.5 min, exclusion list size of 30, and exclusion duration of 3 min. The mass spectrometer was calibrated to meet the resolution specification (10000 at 400 m/z) before sample analysis to ensure instrument the best performance. All MS/MS spectra were searched against the human IPI protein database (version 3.22), which included HBV proteins using the SEQUEST algorithm on Bioworks software (version 3.1) for peptide and protein identifications. Searching results were assembled using BuildSummary software and filtered with the parameters of ΔCn cutoff value ≥0.08 and Xcorr scores ≥1.5 for singly charged, ≥2.5 for doubly charged, and ≥3.5 for triply charged. The DAVID database20,21 was used for functional annotation of identified proteins.

Nuclear Protein Extraction

HBx-TAP-HepG2 and TAP-HepG2 stable cell lines were cultured for 24 h and then treated with 5 μM camptothecin for another 24 h, with control not induced. Cytoplasmic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer's protocol. Proteins were quantified using Pierce BCA Protein Assay (Pierce) according to the manufacturer's instructions. Western Blotting

Approximate 15 μg proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Whatman, Kent, United Kingdom). Membranes were blocked for 2 h with 5% nonfat dry milk in TBST at room temperature and then incubated overnight with different primary antibodies at 4 °C. After they were washed with TBST, the membranes were incubated for 1 h with corresponding horseradish peroxidase (HRP) labeled second antibody at room temperature. Blots were detected by chemiluminescence with SuperSignal West Dura Extended Duration Substrate (Pierce). The following antibodies were used: anti-Flag (1:10000, Sigma), anti-Myc (1:2000, Tiangen), anti-AIF (1:3000, Sigma), anti-β tubulin (1:5000, Sigma), antilamin B (1:2000, Santa Cruz, CA), goat antimouse (1:10000, Cell Signaling, Danvers, MA), goat antirabbit (1:10000, Cell Signaling), and rabbit antigoat (1:5000, Tiangen). Fluorescence-Activated Cell Sorting (FACS) Analysis of Apoptosis

Localization Analysis

HBx-TAP-HepG2.2.15 and TAP-HepG2.2.15 stable cell lines were treated with 5 μM camptothecin (CPT) for 24 h, stained by PI and YO-PRO-1 according to the manufacturer's protocol using Vybrant Apoptosis Assay Kit #4 (Molecular Probes, Eugene, OR), and analyzed by FACS (BD FACSAria II, Franklin Lakes, NJ). HBx-TAP-HepG2 and TAP-HepG2 stable cell lines were seeded in a six-well plate the day before transfection. AIF-EGFP/AMID-EGFP or EGFP expression vectors were transfected into both cell lines, respectively. At 24 h after transfection, 5 μM CPT was added to induce apoptosis for 24 h. Cells were then collected and fixed in 0.5% paraformaldehyde for 1 h at room temperature, followed by fixed in 70% ethanol overnight at 4 °C. Finally, cells were incubated in 0.1 g/L RNAase and 0.1 g/L propidium iodide (PI) for 30 min at room temperature and analyzed by FACS. The percentage of cell death was measured in EGFP-labeled cells using two-color flow cytometry.

HBx-DsRed and AIF-EGFP/AMID-EGFP expression vectors were transiently cotransfected into HepG2.2.15 and HepG2 cells using Lipofectamine 2000, with DsRed and EGFP expression vectors as controls, respectively. At 48 h posttransfection, cells were washed three times with ice cold phosphate-buffered saline (PBS) and fixed in 10% formalin for 30 min at room temperature. The slides were mounted with Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent images of cells were captured by confocal microscopy (LSM 710, Carl Zeiss, Jena, Germany). In experiments intended to study the change of the cellular location of AIF during apoptosis, one more step was added: cells were treated with camptothecin (5 μM 20 h Sigma) at 24 h after transfection. Coimmunoprecipitation (Co-IP)

HBx-Flag and AIF-Myc/AMID-myc expression vectors were transiently cotransfected into HepG2.2.15 cells using Lipofectamine 2000, with Flag and Myc tags expression vectors as controls, respectively. At 48 h post-transfection, cells were washed three times with ice cold PBS, collected by scraping, and incubated with IPP150 with freshly added 1 mM PMSF, 2 mM DTT, and completely EDTA-free Protease Inhibitor (Roche) on ice for 15 min. The cell lysates were then centrifuged at 10000g for 10 min at 4 °C. The supernatants were collected and rotated with the indicated antibodies (antiFlag 1:400, Sigma; anti-Myc 1:100, Tiangen, Beijing, China) and 30 μL of protein A/G-Sepharose (Amersham Biosciences,

Chromatin Condensation Frequency Analysis

HBx-TAP-HepG2.2.15, TAP-HepG2.2.15, HBx-TAP-HepG2, and TAP-HepG2 stable cell lines were cultured on glass slides for 24 h. Then, 5 μM CPT was added to induce apoptosis for 24 h, while control groups were not induced. Cells were then rinsed three times in PBS and fixed in 10% formalin for 30 min at room temperature. The slides were mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories) and observed by Zeiss LSM 710 Laser Scanning Microscopy. To determine the frequency of chromatin condensation, at least 27 different fields were counted. 4805

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Figure 1. Proteins potentially bound to HBx and their functional classification. (A) Overexpression of HBx in HepG2.2.15 cells. Total proteins from HBx-TAP-HepG2.2.15 and TAP-HepG2.2.15 stable cell lines were detected by Western blotting with rabbit IgG, which binds to the TAP tag. (B) The proteins potentially bound to HBx. The interactions were identified by mass spectrometry. Each ellipse or rectangle represents one protein. Each line indicates a protein−protein interaction. Solid lines indicate previously reported protein−protein interactions. Dashed lines indicate newly identified protein−protein interactions. The HBx−AIF and HBx−AMID interactions are two of the newly discovered interactions. (C) Gene ontology of the newly identified proteins. Biological processes at p < 0.05 are shown. The apoptosis process had the most significant p value. (D) Proteins enriched in apoptosis by DAVID.

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performed using the TAP/MS method.17 The TAP purifies proteins first by ProtA-IgG affinity capture and then releases proteins by TEV cleavage, which avoids use of denaturing low pH conditions, and second by CBP-calmodulin affinity capture, again affecting release under mild conditions by sequestering

RESULTS AND DISCUSSION

Identification of HBx−Cell Protein Complexes

A screen to purify and identify the cellular protein partners bound by HBx in HBV-producing HepG2.2.15 cells was 4806

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Figure 2. HBx interactions with AIF and AMID. (A) HepG2.2.15 cells were cotransfected with AIF-Myc and HBx-Flag or Flag expression vectors. Cell lysates were immunoprecipitated with anti-Flag antibody followed by Western blotting with indicated antibodies. (B) HepG2.2.15 cells were cotransfected with HBx-Flag and AIF-Myc or Myc expression vectors. Cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with indicated antibodies. (C and D) The same experiments in panels A and B were performed with AMID-Myc substitute for AIFMyc.

calcium with EGTA.18 The expressions of HBx-TAP and TAPtag were confirmed by Western blotting (Figure 1A). Proteins were then identified in the purified complexes from HBx-TAPHepG2.2.15 cells by MS, excluding the proteins also identified in the TAP-HepG2.2.15 cells as background (Figure 1B). Forty-nine proteins were identified by this analysis, with four proteins (DDB1, TFIIB, HSP60, and HSP70-1) (solid lines, Figure 1B) previously known to be associated with HBx.22−25 It has been reported that HBx can modulate cellular transcription through interactions with TFIIB25−28 and target nucleotide excision repair through a direct binding to DDB1.22 Forty-five previously unreported proteins were newly identified as potential interactors with HBx (dashed lines, Figure 1B). Detailed information for identified proteins is shown in Table 1 in the Supporting Information, and the MS/MS spectra of the proteins identified by only one peptide are shown in Figure 1 in the Supporting Information. It has been previously suggested that the TAP method has several advantages for investigation of multiprotein complexes: it is believed that to avoid the interference that may occur using antibody-based enrichment methods, the presence of high affinity binders in the TAP-tag allows for the enrichment of cell proteins present in low levels, which might be missed by other methods.17 It has also been suggested that the two-step TAP method may identify fewer false positives than single-step purification protocols.18 In addition, the assay examines HepG2.2.15 cells, providing a contrast in the repertoire of

HBx-interacting cellular proteins present during HBV infection to those normal cell proteins found without HBV. However, there are yet other proteins previously reported to associate with HBx, which were not found in our analysis, such as p53, cFLIP, and HBXIP. It might be due to several reasons; the second step of TAP purification uses EGTA to remove calcium and release the protein complex. Although the release conditions are mild, protein interactions requiring calcium may be disrupted, or the TAP-tag may impair some HBx to protein interactions and prevent detection of the interacting protein by this method. We used DAVID Gene Ontology software20,21 to provide a systematic overview of the biological processes in which the proteins that interact with HBx are identified, to provide insights into the potential functions of HBx in HBV-infected hepatoma cells. The result showed that members of “regulation of apoptosis” pathway were the most enriched (p < 0.001) (Figure 1C). Enrichment of this pathway was not unexpected, as HBx has important roles in apoptosis during HBV pathogenesis. Ten proteins involved in apoptosis regulation were enriched, AIF, PECR, AMID, ALB, TUBB2C, HSP70-1, HSPA5, HSP60, RPS3, and HSPA9. Five proteins, ALB, HSP70-1, HSPA5, HSP60, and HSPA9, were categorized as functional negative regulators of apoptosis, whereas the other five, AIF, AMID, TUBB2C, HSP60, and RPS3, were categorized as functional positive regulators of apoptosis, with HSP60 included in both groups (Figure 1D). Three of these 4807

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Figure 3. HBx colocalized with AIF or AMID. (A) HepG2.2.15 cells were cotransfected with the following expression vectors: HBx-DsRed and AIFEGFP (panel 1), DsRed and AIF-EGFP (panel 2), HBx-DsRed and AMID-EGFP (panel 3), DsRed and AMID-EGFP (panel 4), and HBx-DsRed and EGFP (panel 5). At 48 h post-transfection, cells were fixed and then imaged by confocal microscopy. DNA was counterstained with DAPI (blue) in all images. The protein-colocalized regions appear orange or yellow. (B) The same experiments were done in HepG2 cells (scale bar, 10 μm).

before and after co-IP to detect interactions, using anti-Myc and anti-Flag antibodies, respectively. Interactions between AIF-Myc or AMID-Myc and HBx-Flag were observed (Figure 2A,C). Importantly, no co-IP was observed in cells expressing AIF-Myc or AMID-Myc and Flag alone, confirming the specificity of each of the interactions between AIF-Myc and AMID-Myc with HBx-Flag (Figure 2A,C). Furthermore, when HepG2.2.15 cells coexpressed HBx-Flag and AIF-Myc, AMIDMyc, or Myc (negative control) expression vectors, the interactions of HBx-Flag with AIF-Myc and HBx-Flag with AMID-Myc were confirmed, and no co-IP was observed in cells coexpressing HBx-Flag and Myc (Figure 2B,D). Taking each of these reciprocal co-IP experiments and control experiments together, the results confirm the specificity of the interaction of HBx with AIF and with AMID.

proteins (HSPA5, HSP60, and RPS3) are known to modulate apoptosis through the regulation of caspase activity (Figure 1D). It is reported that RPS3 induced apoptosis through activation of caspase-3 and -8,29 and members of the HSP70 family (HSP70-1, HSPA5, and HSPA9) and HSP60 were involved in apoptosis through interactions with caspases, HIV Vpr, or p53,30−33 while albumin (ALB) protects cells from apoptosis through its role in preventing mitochondrial depolarization.34,35 We can conclude from the known functional activities of these proteins that most would regulate apoptosis through the caspase-dependent pathway. However, AIF and its partial homologue AMID are well-known key mediators of the caspase-independent pathway of apoptosis. 10,11,13,14 This finding indicated that the potential interactions between HBx and AIF or AMID may contribute to the processes or regulation of apoptosis and stimulated us to validate their interactions and to investigate the physiological effects.

HBx Colocalizes with AIF and AMID

The cellular localization of HBx, AIF, and AMID in HepG2.2.15 and HepG2 cells was then investigated by fluorescent confocal microscopy. HepG2.2.15 and HepG2 cells were transiently transfected with HBx-DsRed and AIFEGFP or AMID-EGFP expression vectors and with DsRed and EGFP expression vectors used as negative controls, respectively. At 48 h post-transfection, the cells were analyzed by confocal microscopy. A majority of HBx-DsRed and AIF-EGFP or AMID-EGFP were colocalized in compartments of HepG2.2.15 and HepG2 cells (Figure 3A, panels 1 and 3,

HBx Interacts with AIF and AMID

HBx and AIF or AMID were overexpressed in HepG2.2.15 cells, and co-IP tests were performed to examine for any prospective interactions. In detail, HepG2.2.15 cells were cotransfected with AIF-Myc or AMID-Myc and HBx-Flag or Flag (negative control) expression vectors. Cells were harvested at 48 h post-transfection, and the lysates were subjected to coIP using anti-Flag antibody. The proteins were Western blotted 4808

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Figure 4. HBx inhibits apoptosis through an interaction with AIF. (A) HBx-TAP-HepG2.2.15 and TAP-HepG2.2.15 stable cell lines were treated with 5 μM CPT for 24 h, stained by PI and YO -PRO-1, and analyzed by FACS, respectively. In the left panel, P4 shows the apoptotic cells, P2 shows the dead cells, and P3 shows the live cells. The right panel presents the relative fold reduction of apoptotic cells in HBx-TAP-HepG2.2.15 stable cells as compared with TAP-HepG2.2.15 stable cells (means ± SDs; n = 6). (B) HBx inhibited the chromatin condensation frequencies. HBxTAP-HepG2.2.15 and TAP-HepG2.2.15 stable cell lines were treated with 5 μM CPT for 24 h and stained by DAPI, respectively. Cells were photographed with microscopy, and the chromatin condensation frequencies were counted (left panel). The same experiments were done in HBxTAP-HepG2 and TAP-HepG2 stable cell lines (right panel) (means ± SDs; n ≥ 27 random fields). (C) HBx-TAP-HepG2 and TAP-HepG2 stable cell lines were transfected with AIF-EGFP (a and b), AMID-EGFP (c and d), and EGFP expression vectors (e and f), respectively. Cells were treated with 5 μM CPT for 24 h and analyzed by FACS, gating on the EGFP positive population. The right panel showed the relative fold-change of apoptotic cells under four conditions (means ± SDs; n = 7) (*p < 0.05, **p < 0.01, and ***p < 0.001).

and B, panels 6 and 8), while DsRed showed a uniform distribution throughout the cell and was not colocated with

either AIF-EGFP (Figure 3, panels 2 and 7) or AMID-EGFP (Figure 3, panels 4 and 9). Furthermore, EGFP also showed a 4809

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Figure 5. HBx inhibited the translocation of AIF from mitochondrion to nucleus but did not inhibit AIF expression. (A) HepG2.2.15 cells were cotransfected with DsRed and AIF-EGFP expression vectors (panel 1) and HBx-DsRed and AIF-EGFP expression vectors (panel 2), respectively. CPT was added to induce apoptosis of HepG2.2.15 cells for 20 h. The cells were imaged using confocal microscopy. (B) The same experiments were done in HepG2 stable cell lines (scale bar, 10 μm). AIF was located in the nucleus and induced the apoptosis of hepatoma cells after CPT treatment for 20 h (panels 1 and 3). However, AIF and HBx were located in the cytoplasm and inhibited the apoptosis of hepatoma cells after CPT treatment for 20 h (panels 2 and 4). (C) HBx-TAP-HepG2 and TAP-HepG2 stable cell lines were treated with or without CPT for 24 h. Nuclear and cytoplasmic proteins were separated. AIF protein was detected by Western blotting. Lamin B and tubulin were used as internal controls. The amount of truncated AIF decreased in the nuclear fraction of HBx-TAP-HepG2 than TAP-HepG2. 4810

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diffuse fluorescence pattern, which indicated that it was not colocalized with HBx-DsRed (Figure 3, panels 5 and 10). Considered together, these results show that HBx is colocalized with both AIF and AMID. This result is reasonable since all of these three proteins have been reported to localize at mitochondrion.10,11,13,36−39 AIF contains the N-terminal mitochondrial leading sequence (MLS) and is imported to mitochondria functioning as an oxidoreductase normally.10,11 Although AMID lack the MLS, it is reported to localize at mitochondria, too.13 HBx is shown to distribute to mitochondria in several studies. For instance, it is reported to interact with HVDAC3 to modulate the transmembrane potential, and two studies searched for the amino acids region targeting to mitochondria. One found aa 54−57, while the other found that cysteine 115 of HBx is important for mitochondria targeting.36−39 According to these researches, it is probably that HBx interacts with AIF and AMID in the mitochondrion. From the fluorescence pictures, we can see that HBx, AIF, and AMID showed granular distribution, which is the feature of mitochondria, as compared to DsRed and EGFP (Figure 3). We then stained mitochondria with Mito Tracker Deep Red FM (Invitrogen), and the results showed that HBx indeed colocalized with AIF/AMID in mitochondria (Figure 2 in the Supporting Information).

during apoptosis to confirm this visual finding. Consistently, the amount of truncated AIF (57 kDa) found in the nuclear fraction of HBx-TAP-HepG2 cells was less than the levels found in TAP-HepG2 cells after CPT treatment, while even lower amounts were detected in the untreated cells (Figure 5C). During normal apoptosis, AIF is translocated from the mitochondrion to the nucleus. However, we did not observe the change of cytoplasmic AIF level (Figure 5C). We suppose that only a small portion of AIF translocated into nucleus during apoptosis. As there is not any AIF in the nucleus normally, it is easy to observe the increasing during apoptosis. On the contrary, because there is a relatively high level of AIF in the cytoplasm, it is difficult to observe a tiny change during apoptosis. Our results suggest that the presence of HBx inhibits the translocation of AIF from the mitochondrion to the nucleus, presumably as a consequence of direct HBx−AIF interaction and, hence, limits the apoptotic process. This effect of HBx on AIF is functionally similar to the cytoplasm sequestration of p53 by HBx, which reportedly also results in the inhibition of cell apoptosis.3 HBx does not have this function with AMID might be due to the sequence or structural differences between AIF and AMID, as AMID does not possess the mitochondrion or nuclear localization sequences found in AIF, and it is reported to be apoptotic when ectopically expressed with a hitherto unknown mechanism.13,14,40 Probably, there is no translocation of AMID during apoptosis, or alternatively, the interaction between HBx and AMID is not involved in apoptosis regulation but in other functions of AMID.41 Further exploration is needed to understand the effects of the interaction between HBx and AMID on cellular processes. Intriguingly, although some investigations have shown that HBx inhibited apoptosis similar to our findings,3,5,42,43 other studies have found that HBx promoted apoptosis.6,7,44 The reason for these different outcomes is not known but likely arises from diverse experimental systems used in different studies. The differences included different cell lines, different apoptosis stimuli, and either the presence or the absence of HBV. We believe that the observed different effects of HBx on apoptosis may also depend in part on the levels of HBx present in the cells. HBx may enhance cell apoptosis through its cytotoxic effects when expressed at high levels, whereas it is nontoxic when it is expressed at the natural endogenous levels. This suggestion is supported by Arzberger and colleagues' work45 in which they showed that a low level of HBx expression did not sensitize cells to apoptosis. Furthermore, they found that apoptosis of infected cells could prevent the release of infectious virus. The inhibition of apoptosis by HBx would be a mechanism supportive of virus proliferation, a general effect of viral infection detected in cells infected by a variety of viruses such as adenovirus, human cytomegalovirus, HPV, and other viruses.46

HBx Suppressed Cell Apoptosis by Inhibition of the Translocation of AIF

We then sought to test whether HBx could regulate apoptosis through interaction with AIF or AMID, as each factor has been reported to play an important role in apoptosis. We first employed flow cytometric analysis with PI and YO -PRO-1 to identify apoptotic cells and could show that HBx inhibited apoptosis induced by CPT in HepG2.2.15 cells (Figure 4A). Similarly, nuclear condensation analysis demonstrated that the number of apoptotic bodies was reduced in both HBx-TAPHepG2.2.15 and HBx-TAP-HepG2 stable cell lines, as compared to their corresponding controls (Figure 4B). HBxTAP-HepG2 and TAP-HepG2 stable cell lines were then transfected with AIF-EGFP, AMID-EGFP, or EGFP expression vectors, respectively, and treated the cells with apoptosis inducer CPT for 24 h post-transfection. Apoptosis was then quantified by FACS after PI staining, gating on the EGFP positive population. The data showed that the overexpression of either AIF or AMID significantly enhanced CPT-induced cell apoptosis (Figure 4C/b,d vs f). However, apoptosis was attenuated when HBx was expressed together with AIF (Figure 4C/a vs b), but this effect was not observed when HBx was expressed together with AMID (Figure 4C/c vs d). Consistent with the results from Figure 4A,B, HepG2 cells overexpressing HBx displayed reduced apoptosis (Figure 4C/e vs f). Collectively, these results suggest that HBx can inhibit apoptosis by a mechanism involving AIF but not directly involving AMID. We then attempted to define the mechanism by which HBx impairs the action of AIF during induced apoptosis. Both HepG2.2.15 and HepG2 cells examined by fluorescent confocal microscopy after CPT treatment for 20 h in the absence of HBx showed that apoptotic bodies were formed and that AIF was located in the nucleus. However, if HBx was also present, AIF was mostly retained in the cytoplasm in both HepG2.2.15 and HepG2 cells (Figure 5A,B). The protein levels of AIF were semiquantified by Western blotting of nuclear extracts taken from HBx-TAP-HepG2 and TAP-HepG2 stable cell lines

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CONCLUSION In the present study, we purified the HBx interactome under mild conditions, and data analysis revealed enrichment of apoptosis-related proteins, including AIF and AMID. Independent molecular study then showed that HBx can repress apoptosis through the caspase-independent apoptosis pathway by the inhibition of the mitochondrion-to-nuclear translocation of AIF, but not AMID. These findings are in addition to the confirmation of HBx regulation of apoptosis via caspasedependent mechanisms. These new findings add significant 4811

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new insights into the complex mechanisms associated with the regulation of HBV-related apoptosis.

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ASSOCIATED CONTENT

S Supporting Information *

Table of information of proteins identified by MS/MS, figure of MS/MS spectra for proteins identified by single peptide, and figure of HBx colocalized with AIF in mitochondria. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-80726786. Fax: 86-10-62773059. E-mail: zhouyx@ tsinghua.edu.cn (Y.Z.). Tel: 86-10-68171208. Fax: 86-1080705155. E-mail: [email protected]. (F.H.). Present Address # The Laboratory of Cell Aging and Cancer Research, Sun YatSen University School of Life Sciences, Guangzhou 510006, China.

Author Contributions ∇

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grants of the National High Technology Program (2006AA020701), the National Natural Science Foundation (30900061/C010803), the International Science & Technology Cooperation Program of China (2011DFB30370), Yuyuan Grant of Tsinghua Universtity (052203009), and Beijing NOVA Program (2011014). We thank Mingwei Liu, Peng Dong, Chunshu Li, Limin Shang, and Lei Zhao (Institute of Epigenetics and Cancer Research, Tsinghua University School of Medicine) for their excellent technical assistance.

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ABBREVIATIONS HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HBsAg, hepatitis B surface antigen; HBx, hepatitis B X protein; co-IP, coimmunoprecipitation; TAP, tandem affinity purification; MS, mass spectrometry; AIF, apoptosis-inducing factor; AMID, AIF-homologous mitochondrion-associated inducer of death; PRG3, p53-responsive gene 3; CPT, camptothecin

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REFERENCES

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