Identification of Intracellular Proteins Associated with the EBV

May 6, 2008 - Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy,. Sahlgrenska University Hospit...
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Identification of Intracellular Proteins Associated with the EBV-Encoded Nuclear Antigen 5 Using an Efficient TAP Procedure and FT-ICR Mass Spectrometry Alma Forsman, Ulla Rüetschi, Josefine Ekholm, and Lars Rymo* Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, Sahlgrenska University Hospital, University of Gothenburg, S-41345 Gothenburg, Sweden Received November 19, 2007

Epstein-Barr virus nuclear antigen 5 (EBNA5) is one of the first viral proteins detected after primary EBV infection and has been shown to be required for efficient transformation of B lymphocytes. EBNA5 is a protein that has many suggested functions but the underlying biology remains to be clarified. To gain further insight into the biological roles of the proposed multifunctional EBNA5, we isolated EBNA5 containing protein complexes using a modified tandem affinity purification (TAP) method and identified the protein components by LC-MS/MS analysis of tryptic digests on a LTQ-FT-ICR mass spectrometer. The modified TAP tag contained a Protein A domain and a StrepTagII sequence separated by two Tobacco Etch Virus protease cleavage sites and was fused to the C-terminus of EBNA5. Our results confirmed the wide applicability of this two-step affinity purification strategy for purification of protein complexes in mammalian cells. A total of 147 novel putative EBNA5 interaction partners were identified, 37 of which were validated with LC-MS/MS in split-tag experiments or in co-immuno precipitates from HEK293 cell extracts. This subgroup included the Bcl2-associated Athanogene 2 (BAG2) co-chaperone involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. These EBNA5 interactors were further verified by co-immunoprecipitations from cell extracts of three EBV-positive lymphoblastoid lines. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 interactors suggests that EBNA5 might have a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Our study also confirms previously identified interactors including HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, and R- and β-tubulin. Keywords: EBNA5 • EBNA-LP • EBNA5 interacting proteins • tandem affinity purification • FT-ICR

Introduction The Epstein-Barr virus (EBV) is the causative agent or cofactor in the etiology of several human malignancies such as Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma (NPC) and lymphoproliferative disorder in posttransplant- and AIDS-patients.1,2 It is a lymphotropic γ-herpes virus infecting more than 90% of the population worldwide. Following acute infection, the virus establishes a life-long latency in resting memory B cells.3 The 172 kb EBV genome contains more than 80 genes that encode structural and regulatory proteins. For efficient immortalization of resting B cells, six EBV-encoded proteins are required, the nuclear proteins Epstein-Barr virus nuclear antigen (EBNA) -1, -2, -3, -5, -6, and the membrane protein LMP1.4 All of the EBNA proteins have been proposed to play a role in the control of * Address correspondence to: Prof. Lars Rymo, Sahlgrenska University Hospital, Department of Clinical Chemistry and Transfusion Medicine, Pav 8:3, S-41345 Gothenburg, Sweden. Tel.: +46 31 342 40 80. E-mail: [email protected]. 10.1021/pr700769e CCC: $40.75

 2008 American Chemical Society

gene expression in the EBV-infected lymphoblastoid cell. The EBNA genes belong to the same transcription unit and the different mRNAs are generated by alternative splicing from a large primary transcript. Only B-lymphocytes can be efficiently infected by EBV, and infection gives rise to cells permissive to virus replication and expression of the EBNA2 and EBNA5 mRNAs. EBNA2 and EBNA5 (also designated as EBNA-LP) are the first virus proteins expressed during the establishment of the virus in the cell. The so far best characterized function of EBNA5 is its cooperation with EBNA2 in the transactivation of the LMP1 promoter.5–7 We have previously shown that EBNA5 can inhibit pre-mRNA cleavage and polyadenylation in a transient transfection system.8 The EBNA5 polypeptide consists of a 66-amino acid multiple-repeat domain encoded by repeating W1 and W2 exons in the major internal repeat (IR1), and a unique 45-amino acid carboxy-terminal encoded by the Y1 and Y2 exons, which are located downstream of the IR1.9,10 Several isoforms of EBNA5 with different numbers of W1W2 repeats generated by alternative splicing can be detected Journal of Proteome Research 2008, 7, 2309–2319 2309 Published on Web 05/06/2008

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during primary infection. Still, most established lymphoblastoid cell lines (LCLs) express one major and a few minor species. EBNA5 contains several phosphorylation sites of which one has been shown to be essential for EBV-induced immortalization of B-lymphocytes and to be involved in the tight association between EBNA5 and the nuclear matrix.12 Several interaction partners of EBNA5 have previously been identified including pRb,13,14 p53,14,15 Hsp70/Hsc70,16–18 Hsp27,3 DNAPKcs,3 R- and β-tubulin,19 Prolyl-4-hydroxylase,19 HA95,19 HAX-1,20,21 hERR-1,22 p14ARF,23 S3a24 and Sp100.25 Multiprotein complexes involving EBNA5 have not been isolated so far. A considerable amount of evidence has accumulated indicating that EBNA5 is a multifunctional protein, although its precise role in EBV biology has yet to be defined. In the present study, we are focusing on the identification of EBNA5 interaction partners in processes that do not necessarily depend on the presence of EBNA2. We are particularly interested in the mechanism underlying the previously described repressor effect of EBNA5.8 Purification of specific protein complexes and identification of their protein components are important strategies to unveil the functions of proteins with known sequence but unknown biological activity. Several approaches have been developed for this purpose, such as twohybrid methods, enzyme assays and tandem affinity purification (TAP) methods followed by mass spectrometric analysis. We have used an improved tandem affinity purification method for purification of proteins interacting with EBNA5 in mammalian cells. With this method, we confirmed a number of previously identified EBNA5 binding partners and also uncovered 147 novel putative protein associations, of which 37 have been validated with immunoprecipitation or split-tag experiments. Among these, Bcl2-associated Athanogene 2 (BAG2), heterogeneous ribonucleoprotein M and 26S Proteasome nonATPase subunit 2 are of particular interest for the elucidation of EBNA5’s biological activity since they are involved in protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformations or target them for degradation. These interactions with EBNA5 have also been verified by immunoprecipitation in extracts of three EBV-positive lymphoblastoid cell lines.

Materials and Methods Plasmid Construction. All constructs made were verified by dideoxy sequencing,26 utilizing the ABI PRISM Big Dye terminator cycle sequencing kit (Applied Biosystems). The EBNA5 expression vector pCI-7(W1W2)Y1Y2, which contains 7 copies of the EBNA5 W1W2 repeat and the unique Y1Y2 sequence, has been described previously.8 The construct pCI-4(W1W2)Y1Y2 (4 copies of the W1W2 repeat and one Y1Y2 sequence) was cloned from the plasmid pSG5-EBNA-LP (a kind gift from Dr. A. Bell, Birmingham).7 The EBNA5 coding sequence was amplified by PCR; an EcoRI endonuclease site was introduced into the upstream primer and an XbaI site into the downstream primer. The amplified fragment was inserted between the EcoRI and XbaI cloning sites of the pCI vector (Promega). An expression vector coding for a fusion protein consisting of the four repeat EBNA5 sequence, a StrepTagII peptide (WSHPQFEK) and one copy of the IgG binding domain of Protein A was constructed. The StrepTagII and the Protein A sequences were separated by two copies of a Tobacco Etch Virus (TEV) protease cleavage site. The coding sequence for EBNA5 was amplified from the pSG5-EBNA5 by PCR and cleaved with EcoRI and NcoI. The StrepTagII sequence and one TEV cleavage site 2310

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Forsman et al. were obtained by hybridization of single-standed oligonucleotides; StrepTagII (5′-AGT AAG CTT TGG AAT ATC CTT TTC GAA CTG CGG GTG GCT CCA GTT TGC CAT GGA TCC TC-3′ and 5′-GAG GAT CCA TGG CAA ACT GGA GCC ACC CGC AGT TCG AAA AGG ATA TCC CAA AGC TTA CT-3′) and TEV site (5′-CCA AAG CTT GAG AAT TTG TAT TTT CAG GGT GAT ATT CCA ACT ACT GCT AGC GA-3′ and 5′-TCG CTA GCA GTA GTT GGA ATA TCA CCC TGA AAA TAC AAA TTC TCA AGC TTT GG3′). The two double-stranded oligonucleotides were cleaved with HindIII, NcoI and NheI and cloned simultaneously with the EBNA5 fragment into the pCI vector (Promega). The second TEV and ProtA sequence was amplified by PCR from the pBS1479 plasmid (Euroscarf), the product was cleaved with NheI and XbaI restriction endonucleases and ligated to the 3′end of the TEV sequence in the above-described plasmid construct, yielding the EBNA5-StrepTAP plasmid. The control plasmid StrepTAP was constructed by PCR amplification of the StrepTagII-TEV-TEV-ProtA sequence from the EBNA5-StrepTAP plasmid. The product was cleaved with EcoRI and XbaI and ligated into the pCI vector. The plasmid coding for HA-tagged BAG2 (pEF-BOS/HA-BAG2) was a kind gift from Dr. S. Hattori. The construct EBNA5-ProtA was generated from the EBNA5StrepTAP construct by cutting out EBNA5 with EcoRI and NcoI; the coding sequence for ProtA was amplified by PCR from the EBNA5-StrepTAP construct and digested with NcoI and XbaI. The two fragments were cloned into an EcoRI and XbaI cleaved pCI vector. For construction of StrepTagII tagged hnRNP M (hnRNP M-Strep), the hnRNP M sequence was amplified by PCR from the plasmid pHCM4 (a kind gift from Dr. M. Swanson) and digestion of the PCR product was performed with SmaI and PstI. The StrepTagII sequence was produced by hydridization of single-standed oligonucleotides and digestion with PstI and NotI. The fragments were ligated into a SmaI and NotI cleaved pCI vector. Cloning of the BAG2-StrepTagII construct was performed in a similar way as the hnRNP M-StrepTag II construct. The BAG2 sequence was amplified by PCR from the pEF-BOS/HA-BAG2 plasmid and digestion of the PCR product was performed with XhoI and KpnI. The StrepTagII sequence was produced by hydridization of single-standed oligonucleotides and digestion with KpnI and SalI. The fragments were ligated into an XhoI and SalI cleaved pCI vector. Cell Culture, Transfection and Immunoassays. HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 100 units/mL penicillin/streptomycin and 100 mM L-glutamine (Invitrogen). Approximately, 20 × 106 cells/175 cm2 cell culture flask were transfected with 20 µg of plasmid and FuGENE6 (Roche Applied Science) according to the manufacturer’s instructions. DG75 cells were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 100 units/mL penicillin/streptomycin. Cells (10 × 106) were transfected with 1.0 pmol plasmid with electroporation as described elsewhere.8 For immunoassay of transfected DG75 cells or the WW1, IB4 and CBC-Rael lymphoblastoid cell lines, 10 × 106 cells were used in each assay. Transiently transfected cells were harvested 72 h posttransfection. The cells were harvested with a cell scraper in 1× PBS and centrifuged at 1000 rpm, 5 min at room temperature. The following antibodies were used for immunoassays: antiEBNA5 (JF186, a kind gift from Dr. M. Masucci, Karolinska Institutet, Stockholm), anti-Hsp70 (Stressgen), anti-Hsc70 (Stressgen), anti-HA High Affinity (Roche), anti-CHIP (Calbiochem), anti-HspBP1 (Transduction Laboratories), anti-hnRNP M (Santa

EBNA5 Multi Protein Complexes Cruz), anti-PSF (Sigma-Aldrich), anti-p54nrb (BD Transduction Laboratories), anti-p68 (Upstate Cell Signaling), anti-BAG2 (a kind gift from Prof. J. Ho¨hfeld, Rheinische Friedrich-WilhelmsUniversity, Bonn), anti-BAG3 (Onkogene), anti-Hsp40 (Stressgen), anti-Hsp60 (Santa Cruz), anti-Hsp70 (Stressgen), antiHsc70 (Stressgen), anti-Hsp90 (Santa Cruz), anti-26S proteasome S2 (Abcam), anti-nucleolin (Abcam), anti-hnRNP A2/B1 (Santa Cruz), anti-hnRNP K (Santa Cruz), anti-R-tubulin (Molecular probes), and anti-β-tubulin (Santa Cruz). For immunoassays, 10 × 106 cells were resuspended in 1 mL of IPP100 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% Triton X-100, and 10% glycerol) supplemented with protease inhibitors (Complete Mini, EDTA-free, Roche Applied Science). The cells were freeze-thawed three times [1 min in N2(l), then 4 min at 37 °C] and left on ice for 30 min. The lysed cells were centrifuged at 13 000 rpm for 15 min at 4 °C. A 250 µL portion of the supernatant was used for one co-immunoprecipitation experiment. The supernatant was precleared with 50 µL of Protein A or G beads (Amersham Bioscience) for 40 min at 4 °C with gentle rocking. The slurry was centrifuged, the cleared supernatant was transferred to a fresh tube, and 2 µg of antibody was added. In control experiments, the antibody was replaced by the same amount of IgG from mouse or rabbit serum. The mixture was incubated for 1.5 h at 4 °C with gentle rocking. The antibody was captured with 75 µL of Protein A or G beads (Amersham Bioscience) for 1 h at 4 °C. The beads were washed with 3 × 1 mL of IPP100 and the captured proteins were released into 50 µL of 2× LDS Sample buffer (Novex, Invitrogen) and incubated at 70 °C for 10 min. Finally, 15 µL of sample was loaded on a SDS-PAGE gel. Immunoblotting was performed using the JF186 anti-EBNA5 antibody. Assessment of the Effect of Affinity Tags on EBNA5 Function. The effect of N- and C-terminal tagging of the EBNA5 protein on the co-activating and repressing functions was investigated in transient transfection experiments using a luciferase reporter plasmid under the control of the LMP1 promoter. DG75 cells27 were transfected with pLRS(-634)Luc and pE∆A6 (EBNA2 expression vector), together with wild-type or TAP variants of EBNA5 expression vectors under conditions described previously.8 Cells were harvested after 48 h and analyzed with the Luciferase Reporter Assay (Promega) according to the manufacturer’s instructions. Immunofluorescence Staining. The cells were grown on coverslips or centrifuged on glass slides in a Cytospin centrifuge at 750g for 3 min. Cells were fixed with methanol/acetone (1: 1) at -20 °C for 20 min and then rehydrated in 1× PBS for 30 min. Antibodies were diluted in blocking buffer (2% BSA, 0.2% Tween-20, 10% glycerol, and 0.05% NaN3 in PBS). The fixed cells were incubated with the anti-EBNA5 JF186 antibody in a moist chamber for 60 min at room temperature, followed by three washes with 1× PBS. The slides were incubated with the secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) for 30 min at room temperature, followed by three 1× PBS washes. The glass slides were mounted with Prolong gold antifade reagent with DAPI (Molecular Probes). Images were collected using an Axioskop 40 (Zeiss) microscope, equipped with ZeissPlan-NEOPLUAR 100×/1.30 oil immersion objective, and recorded on a PC computer equipped with the Cytovision software (Applied Imaging). Digital images were assembled using Adobe Photoshop software. Purification of the EBNA5-StrepTAP-Protein Complexes. After harvest, the cells were resuspended in 1 mL/20 × 106 cells of IPP100 with protease inhibitors. The cells were freeze-thawed

research articles three times [1 min in N2(l), then 4 min at 37 °C] and left on ice for 30 min. The lysed cells were centrifuged at 13 000 rpm, 15 min at 4 °C, and the supernatant was incubated with 250 µL of IgG-beads (Amersham), prewashed with IPP100, for 3 h at 4 °C with gentle agitation. After incubation, the beads were washed with 5 mL of IPP100 and 5 mL of 10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, and 10% glycerol (TEV cleavage buffer). The beads were resuspended in 700 µL of TEV cleavage buffer and 70 units of TEV protease were added. The beads were incubated at 4 °C overnight with gentle rocking. After TEV cleavage, the supernatant was added to a 200 µL prepacked column with StrepTactin matrix (IBA GmBH), and the column was washed with 10 mL of TEV cleavage buffer. The protein complexes were eluted from the StrepTactin matrix with TEV cleavage buffer containing 5 mM D-desthiobiotin. Proteins in the eluate were TCA-precipitated and the pellet was dissolved in 1× LDS Sample buffer (Novex, Invitrogen), heated at 70 °C for 10 min, and loaded onto a SDS-PAGE gel (Invitrogen). Proteins were visualized by silver (SilverQuest, Invitrogen) or Coomassie-Blue staining (Colloidal Blue, Invitrogen). Sample Preparation and Mass Spectrometry. Each of the protein-containing lanes of the 1D SDS-PAGE gels were excised and divided into 12-24 equally sized gel pieces. Individual gel pieces were subjected to in-gel digestion with trypsin resulting in 24 samples originating from the EBNA5-StrepTap eluates and 12 samples from the StrepTap control eluate. For trypsin digestion, the method described by Davidsson et al.28 was applied with some modifications. Gel pieces were washed in 400 µL of water/acetonitrile (ACN) 1:1 for 2 × 15 min and then dehydrated in 200 µL of ACN. The gel pieces were air-dried for ∼15 min and rehydrated in 20 µL of 10 mM DTT, 0.1 M NH4HCO3, and reduction of disulfide bonds was performed at 56 °C for 45 min. The supernatant was discarded and cysteine residues were modified to S-carboxyamidomethylcysteine in 20 µL of 55 mM iodoacetamide, 0.1 M NH4HCO3 in the dark at room temperature for 30 min. After washing with 200 µL of 0.1 M NH4HCO3/ACN (1:1) for 15 min followed by 200 µL of ACN, the gel pieces were air-dried for ∼15 min, rehydrated in 10 µL of chilled 50 mM NH4HCO3 and 12.5 ng/µL trypsin, and incubated at 37 °C overnight. The supernatant was collected and peptides were extracted from the gel twice with 30 µL of 50 mM NH4HCO3/ACN (1:1) followed by 30 µL of 5% formic acid/ACN (1:1). The combined extracts were evaporated to dryness in a vacuum centrifuge. Prior to mass spectrometric analysis, peptides were redissolved in 10 µL of 0.1% formic acid. Online peptide separation was performed on a 150 × 0.075 mm fused silica column (Zorbax 300-SB-C18, Agilent technologies) with a 50 min gradient from 0 to 50% ACN and 0.1% formic acid at a flow rate of 200-300 nL/min. A tapered fused silica was used as an emitter. Mass analyses were performed with a hybrid linear ion trap FT-ICR mass spectrometer equipped with a 7T ICR magnet (LTQ-FT; Thermo Electron, Bremen, Germany). The mass spectrometer was operated in a datadependent mode to automatically switch between MS and MS/ MS acquisition. Survey MS spectra (from m/z 350 to 1500) were acquired in the FT-ICR, and the three most intense ions in each FT scan were fragmented and analyzed in the linear ion trap (LTQ). Data Analysis and Protein Identification. Proteins were identified by automated database searching (MASCOT, http:// www.matrix-science.com) of all MS/MS spectra using the Swiss-Prot database. LTQ .raw files were converted to .dta files Journal of Proteome Research • Vol. 7, No. 6, 2008 2311

research articles by the XCalibur software utility extract_msn (Thermo Fisher Scientific). Mascot search parameters were set as follows: MS accuracy, 15 ppm; MS/MS accuracy, 0.5 Da; one missed cleavage allowed; variable carbamidomethyl modification of cystine and variable oxidation of methionine and all entries of the Swiss-Prot database were searched. To get a measure of the false positive rate of the database searches, a randomized version of the Swiss-Prot database was constructed using the decoy.pl script (Matrix science, http://www.matrix-science. com). The search parameters used to search the randomized database were the same as described above. Size Exclusion Chromatography. Whole cell lysates were prepared by resuspending the harvested cells in 1 mL/20 × 106 cells of IPP100 with protease inhibitors followed by three cycles of freeze-thawing [1 min in N2(l), then 4 min at 37 °C]. The cell extracts were centrifuged in two steps, at 13 000 rpm for 15 min at 4 °C followed by 100 000g for 30 min at 4 °C. The resulting supernatants were subjected to gel filtration using a Superose 6 (GE Healthcare) column equilibrated in 10 mM TrisHCl, pH 8.0, and 100 mM NaCl. In parallel, eluates from the affinity purification were run on the same gel filtration system. Collected fractions were precipitated with trichloroactetic acid and analyzed with immunoblotting, or subjected to tryptic digestion and identification of proteins with LC-MS/MS.

Results Generation of a Modified Tandem Affinity Purification Tag for Efficient Capture of EBNA5-Interacting Intracellular Proteins. In the light of recent findings that functional units of the cell are not single proteins but well-structured complexes/ machineries composed of multiple proteins, we attempt to gain further insight into the roles of EBNA5 in EBV infection and cell transformation by isolation of EBNA5-containing protein complexes and identification of their components. In the original version of the Tandem Affinity Purification (TAP) method described by Rigaut et al.,29 the TAP construct consisted of two IgG-binding units from Protein A, a cleavage site for the tobacco etch virus (TEV) protease, and a calmodulin binding peptide. This procedure was very successfully utilized in yeast.30,31 However, in an initial series of experiments using the original TAP-tag constructs in the EBV negative lymphoid B-cell line DG75, we obtained high background levels. This, in combination with the low transfection efficiency of lymphoid cells, made us change the target cell type to HEK293 epithelial cells, which have been successfully used for TAP analyses of EBV oriP-binding proteins.32 In addition, we modified the affinity tag by replacing the calmodulin binding peptide with one copy of the StrepTagII peptide33,34 followed by two TEV protease cleavage sites and the IgG-binding Protein A domains. This change significantly improved the sensitivity of the method. A similar modification of the TAP tag was reported during the course of our investigation.35 The modified tag was cloned downstream of the EBNA5 open reading frame in a pCI expression vector (Figure 1). In a previous study, we have shown that EBNA5, expressed at high but biologically relevant levels, in addition to being a co-activator of EBNA2, functions as a repressor of reporter gene expression in the absence of EBNA2.8 A major goal of the present investigation was to increase our knowledge of the mechanism underlying the EBNA2-independent activity. The ability of the EBNA5-TAP protein to co-activate EBNA2-induced activation of the LMP1 promoter and to repress gene expression as a function of the position of the TAP tag were assessed by reporter gene assays. 2312

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Figure 1. Schematic representation of constructs used in the StrepTAP affinity purification. (I) The EBNA5-StrepTAP construct used consists of EBNA5 with 4 copies of the W1W2 repeat and the unique Y1Y2 domain, flanked by the StrepTagII affinity tag and the Protein A domain from Staphylococcus aureus, separated by two TEV protease cleavage sites. (II) In the control construct, the EBNA5 domain is removed, while the StrepTagII, TEVx2 and the Protein A domains from EBNA5-StrepTAP remain. (III-V) Representation of the constructs used in the spilt-tag experiments. (III) EBNA5 with the C-terminal Protein A domain separated with two TEV protease cleavage sites; (IV and V) BAG2 and hnRNP M N-terminal of the StrepTagII affinity tag.

EBV negative DG75 lymphoid B cells were transiently transfected with plasmids coding for either wild-type, N-terminally or C-terminally TAP-tagged EBNA5 together with an EBNA2 expression vector and a luciferase reporter plasmid under control of the LMP1 promoter. The level of reporter activity induced by the tagged EBNA5 was similar to that obtained with the untagged EBNA5 construct (Figure 2A). Furthermore, the C-terminally tagged EBNA5-TAP fusion protein repressed luciferase expression as efficiently as the untagged EBNA5 protein, whereas the N-terminally tagged version no longer had a significant inhibitory effect (Figure 2A). On the basis of these results, the modified EBNA5-StrepTAP was designed to carry a C-terminal tag, activation and repression characteristics were verified with the modified tag (Figure 2B), and this construct was used in all experiments described ahead. In addition, we compared the intracellular localization pattern of EBNA5StrepTAP in transiently transfected HEK293 with that of the endogenous EBNA5 in three EBV immortalized lymphoblastoid B-cells (Figure 3). At our level of resolution, the TAP-modified EBNA5 in HEK293 cells showed a granular pattern of EBNA5 in the nucleus similar to that in the LCLs. Purification of Protein Complexes Using EBNA5 as the Bait. HEK293 cells were transiently transfected with EBNA5StrepTAP, and the EBNA5-StrepTAP cellular protein complexes were purified by two consecutive affinity chromatography steps under native conditions. Cell lysates were applied to an IgGSepharose resin and bound proteins were eluted by proteolytic cleavage of the StrepTAP tag at the TEV protease sites. In a parallel experiment, the same procedure was performed with the control construct expressing the StrepTAP tag alone. Eluates from the proteolytic cleavage on the IgG resin were incubated with a StrepTactin matrix and proteins bound to the StrepTactin matrix were eluted by specific competition with Ddesthiobiotin. Proteins co-purifying with EBNA5-StrepTAP or StrepTAP were separated by SDS-PAGE (Figure 4). Each of the protein-containing lanes of the gel were excised and divided into 12-24 equally sized gel pieces. Individual gel pieces were subjected to in-gel digestion with trypsin resulting in one set

EBNA5 Multi Protein Complexes

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Figure 3. Immunofluorescent staining of EBNA5 shows granular nucleoplasmic distribution in the lymphoblastoid cell lines WW1LCL, IB4, and CBC-Rael, as well as in HEK293 cells transiently transfected with the EBNA5-StrepTAP construct. Green, EBNA5; blue, DNA-staining; red, cytoplasmic staining. Figure 2. Assessment of the influence of affinity tags on EBNA5 function. The effect of N- and C-terminal tagging of the EBNA5 protein on the co-activating and repressing functions was investigated in transient transfection experiments using a luciferase reporter plasmid under the control of the LMP1 promoter. DG75 cells27 were transfected with pLRS(-634)Luc and pE∆A6 (EBNA2 expression vector), together with wild-type or TAP variants of EBNA5 expression vectors.8 Cells were harvested after 48 h and analyzed with the Luciferase Reporter Assay (Promega). Activation was expressed as luciferase activity obtained after addition of EBNA5 variants relative to the reporter activity with EBNA2 alone. Panel A shows the results for the Nand C-terminally TAP-tagged EBNA5, panel B shows the results for the C-terminally StrepTAP-tagged EBNA5. RLU, relative luciferase activity.

of samples originating from the EBNA5-StrepTAP eluate and one set from the StrepTAP control eluate. MS/MS Identifications and Minimization of False Positive Results. Identification of the proteins co-purified with EBNA5-StrepTAP or StrepTAP was performed by LC-MS/MS analysis of tryptic digests on a Hybrid Linear Ion Trap (LTQ)FT-ICR mass spectrometer. LC separations were performed on a nanoscale reversed-phase column and peptides were analyzed as they eluted off the column into the nanospray ionization probe of the spectrometer. For each MS full scan in the ICR cell, the three most abundant peptides were selected for fragmentation and MS/MS analysis in the linear ion trap (LTQ) of the mass spectrometer. All acquired MS/MS spectra were searched against the complete Swiss-Prot protein sequence database. To address the issue of false positive protein identifications, a randomized version of the Swiss-Prot database was constructed and used for parallel data searches. For each peptide matching a query MS/MS spectrum, MASCOT calculates a probability-based “Ion Score”, which is defined as the logarithm of the probability that the observed match between experimental data and the database sequence is a random

Figure 4. SDS-PAGE separation of EBNA5 containing protein complexes. StrepTAP purified eluates were separated on a denaturing 4-12% Bis-Tris ready gel and silver-stained. Lane 1 (EBNA5), protein complexes co-purified with EBNA5-StrepTAP; Lane 2 (Control), proteins purified with the StrepTAP control.

event. Knowing the size of the sequence database being searched, it becomes possible to provide an objective measure of the significance of a result. MASCOT also reports an expectation value; it can be derived directly from the score and significance threshold values, and it gives the number of times you could expect to get this score or better by chance. In a MS/MS search, the protein score is derived from the scores of the individual peptide matches. It is important to keep in mind that the protein score in a MS/MS search is not statistically rigorous; it provides a way to rank the protein hits. In this study for a positive protein hit to be selected, the following criteria had to be met. Minimum two peptide hits were required, one at a 95% confidence level and one at a 99% confidence level; at least one of the peptides had to be the top scoring match for the spectrum and the protein was the highest scoring in Journal of Proteome Research • Vol. 7, No. 6, 2008 2313

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Figure 5. Validation of protein-protein interactions. (A) Western blot on eluates from the two step StrepTAP purification. The analysis was performed with antibodies directed against Hsp70, Hsc70, and R- and β-tubulin, to verify the abundance of the proteins in the EBNA5 eluate; [E5] indicate eluate form EBNA5-StrepTAP, and [C] eluate from StrepTAP. (B) Co-immunoprecipitation on whole cell lysates from HEK293 cells transfected with either pCI-EBNA5 [E5] or pCI [C] were performed with various antibodies as depicted in the figure. The presence of the EBNA5 protein in the precipitated complexes was detected with Western blot against EBNA5. The arrow shows the location of the specific EBNA5 band, the arrowhead shows cross-reactivity with the heavy chain of antibodies used for immunoprecipitation. (C) Co-immunoprecipitation on whole cell lysates from DG75 cells transfected with either pCI-EBNA5 [E5] or pCI [C] were performed with various antibodies as depicted in the figure. (D) Co-immunoprecipitation on whole cell lysates from EBVimmortalized lymphoblastoid cell lines. [E5] indicates presence of EBNA5 in the pulldown experiment and [IgG] indicates pulldown with normal IgG from mouse, rabbit or goat.

which the peptide was found (“bold-red”). Using these criteria on the results from database search with the EBNA5-StrepTAP MS/MS data, 170 proteins were identified as EBNA5 interactors (Supplementary Table 1). The same selection criteria applied to results with the StrepTAP control generated a list of 29 nonspecific protein interactors and 18 of them were present in the EBNA5 sample (Supplementary Table 2). Applying the selection criteria to the results of the reversed database search produced no positive protein hits in any of the data sets. Further data reduction was performed by subtracting the nonspecific interactors from the list of proteins interacting with EBNA5-StrepTAP (Supplementary Table 3). Among the nonspecific interactors co-purifying with both EBNA5-StrepTAP and the Strep-TAP control were Hsp70, Hsc70, and tubulin R and β. These proteins have been reported to be specific interactors of EBNA5 by other groups and their interaction with EBNA5 has been studied in detail. Kieff and co-workers showed a specific interaction between EBNA5 and Hsp70 by immunoprecipitation with an EBNA5-specific antibody.17 Precipitated proteins were identified by Matrix Assisted Laser Desorption (MALDI) mass spectrometry and peptide sequencing.17 A similar approach was used to identify proteins interacting with 2314

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a Flag-epitope tagged EBNA5.19 Both Hsp70 and Hsc70, and tubulin R and β were among the proteins identified as EBNA5 interactors. Furthermore, a cooperation of Hsp70 and EBNA5 in regulating EBNA2 driven transcription from the LMP1 promoter has recently been described.25 A survey of our mass spectrometry data and MASCOT results indicated that the amounts of Hsp70, Hsc70 and tubulin R and β were much higher in the EBNA5-containing samples compared to the controls. Since mass spectrometry is not an inherently quantitative method, we used immunoblotting to measure the amounts of Hsp70, Hsc70, and tubulin R and β in eluates from the StrepTAP affinity purifications. The results showed a distinct signal in the EBNA5-StrepTAP eluate, while the amount of Hsp70, Hsc70, and tubulin R and β in the StrepTAP control eluate was below the detection limit (Figure 5A). We conclude that the interaction between EBNA5StrepTAP and Hsp70, Hsc70, and tubulin R and β is specific. However, small amounts of these highly abundant proteins copurified with the StrepTAP control in a presumably nonspecific manner. A complete compilation of the proteins determined to interact with EBNA5 is given in Supplementary Table 4. A total of 147 novel EBNA5-associated factors were uncovered

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EBNA5 Multi Protein Complexes a

Table 1. Validation of Specific Protein Interactors of EBNA5 biological function

protein

Protein folding and degradation BAG family molecular chaperone regulator 2 BAG family molecular chaperone regulator 3 Heat shock protein 40 Heat shock protein 60 Heat shock protein 70 Heat shock cognate 70 Heat shock protein 90 Proteasome 26S non-ATPase regulatory subunit 2 Ubiquitin Pre-mRNA processing Heterogeneous nuclear ribonucleoprotein A/B Heterogeneous nuclear ribonucleoprotein A1 Heterogeneous nuclear ribonucleoprotein K Heterogeneous nuclear ribonucleoprotein M Heterogeneous nuclear ribonucleoprotein Q Heterogenous nuclear ribonucleoprotein U p54nrb Splicing factor, proline- and glutamine-rich (PSF) Ribosomal proteins 40S ribosomal protein S4 40S ribosomal protein S8 40S ribosomal protein S13 40S ribosomal protein S16 40S ribosomal protein S18 40S ribosomal protein S23 40S ribosomal protein S25 60S ribosomal protein L11 60S ribosomal protein L12 Miscellaneous Aspartyl-tRNA synthetase ATP-dependent DNA helicase II 80 kDa ATP-dependent RNA helicase A Bifunctional aminoacyl-tRNA synthetase Glutaminyl-tRNA synthetase Interleukin enhancer-binding factor Isoleucyl-tRNA synthetase Nucleolin Propionyl Coenzyme A carboxylase Tubulin-R Tubulin-β a

O95816 Q9JLV1 P25685 P10809 P08107 P11142 P07900 Q13200 P62988 Q99020 P09651 P61978 P52272 O60506 Q00839 Q15233 P23246 Q6PBC4 P62241 P62277 P62249 P62269 P62266 Q6Q311 P27635 P23358 P14868 P13010 Q08211 P07814 P47897 Q12906 P41252 P19338 P05165 P68363 P07437

co-immunoprecipitation

BAG2

hnRNP M

• • • • • • • • nt • nt • • nt nt • • nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt nt • nt • •

•

•

• •

• •

• • •

• • •

• • •

•

• • • •

•

• • • • •

• • • • • • • • • • • • • •

• • • •

• • • •

Circles (•), detected with LC-MS/MS in split-tag experiments or positive outcome in co-immunoprecipitation experiments; nt, not tested.

including the Bcl2-associated Athanogene 2 (BAG2) cochaperone, which is involved in protein folding and renaturation, the 26S proteasome subunit 2 involved in regulation of ubiquitin/proteasome protein degradation, and the heterogeneous ribonucleoprotein M (hnRNP M) involved in pre-mRNA processing. The combination of the Hsp70, Hsc70, BAG2 and 26S proteasome subunit 2 partners is interesting and compatible with the notion that EBNA5 has a functional relationship with protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. Consistent with previous reports, HA95, Hsp70, Hsc70, Hsp27, HAX-1, Prolyl 4-hydroxylase, S3a, R- and β-tubulin, were also present in the eluates from EBNA5-StrepTAP affinity purification. Validation of Novel Protein Interactions. Interactions detected with the EBNA5-StrepTAP tag should be confirmed with independent methods. We validated a number of interactions by immunoprecipitation of proteins from HEK293 cells transiently transfected with wild-type EBNA5 or a noncoding control plasmid (Figure 5B). Protein complexes were pulled down from cell extracts using antibodies specific for the BAG2, BAG3, Hsp40, Hsp70, Hsc70, Hsp90, 26S proteasome subunit 2, nucleolin, hnRNP M, hnRNP A/B, hnRNP K, tubulin R, tubulin β, p54nrb, p68 and PSF proteins. Immune precipitates

were analyzed by Western immunoblotting using an antiEBNA5 antibody. EBNA5 bands were detected in all blots obtained with wild-type EBNA5 samples but not in any derived from control samples. Thus, the results strongly supported the notion that EBNA5 forms complexes with the immunoprecipitaded proteins (Figure 5B, Table 1). No specific bands were observed in control experiments where the immunoprecipitations had been performed with the IgG fraction from normal mouse or rabbit serum. Some interactions identified with the EBNA5-StrepTAP tag were validated by detection with LC-MS/ MS in split-tag experiments using BAG2 or hnRMP M as baits (Table 1). The majority of the 37 validated EBNA5 interactors could be assigned to one of three groups according to function: protein folding and degradation, pre-mRNA processing, and ribosomal proteins. To increase the probability of a biological significance of these EBNA5 interactions and to demonstrate that they also existed in the context of lymphoid cells, we selected three interactors with clear functional implications, BAG2, 26S proteasome subunit 2 and hnRNP M, for further analysis at high (overexpression) or low (endogenous) EBNA5 levels. Immunoprecipitations were performed using BAG2, 26S proteasome subunit 2 and hnRNP M antibodies and extracts of EBV negative DG75 cells transiently transfected with EBNA5 (Figure Journal of Proteome Research • Vol. 7, No. 6, 2008 2315

research articles 5C), and extracts of the EBV positive lymphoblastoid cell lines WW1-LCL, IB4 and CBC-Rael, respectively (Figure 5D). Immune precipitates were analyzed by immunoblotting using the antiEBNA5 JF186 antibody. EBNA5 bands were detected in all lanes where transfected DG75 cells or EBV positive LCLs had been analyzed. None of the lanes that contained DG75 cells transfected with empty vector DNA or LCL control cells immunoprecipitated with the IgG fraction of nonimmune sera displayed EBNA5 specific bands. Together, the results support the notion that EBNA5 complexes, identified in this communication by affinity-purification combined with MS and validated by immunoprecipitation of extracts of HEK293 cells and lymphoblastoid cell lines, represent true and meaningful interactions of functional significans. EBNA5 Complex Isoforms. Modularity has been proposed as a general principle for the molecular architecture of living systems. Functional units are not single proteins but wellstructured complexes composed of multiple proteins.36 In fact, half of the yeast proteome was recently reported to be in complexes.37,38 Substantial protein sharing between complexes was observed suggesting that proteins can be combined in multiple ways to diversify the functional repertoire within cells. To throw some light on the question whether the EBNA5 complex obtained with the method described above contained several functional isoforms, we employed a split-tag purification strategy for the isolation of the complexes. To this end, Protein A was fused as an affinity tag (Figure 1) to EBNA5 and the StrepTagII tag was fused to either of the hnRNP M or BAG2 proteins, regularly identified with our two-step TAP procedure as components of the EBNA5 complex but representing two different cellular machineries. HnRNP M is an hnRNP protein with an established role in the processing of pre-mRNA and BAG2 is an Hsp70/Hsc70 binding protein present in the eukaryotic cytosol with a regulatory function in the chaperone cycle. The purification procedure was similar to that described for the two-step method, except that in the second affinity purification step the selection procedure was designed so as to obtain complex fractions that contained either the tagged hnRNP M or the tagged BAG2 proteins. The protein components of the purified complexes were separated by SDS-PAGE, and the individual lanes were excised, cut into slices, subjected to in-gel tryptic digestion and analyzed as described in Materials and Methods. The results showed that about 80% of the proteins identified with the hnRNP M and BAG2 baits were the same and similar to those identified with our standard twostep TAP tag procedure (Supplementary Table 4). The size and composition of the EBNA5-StrepTAP complexes were investigated further by Sepharose 6 gel filtration of the purified EBNA5 complexes under native conditions. Determination of the elution patterns of the EBNA5, Hsp70, Hsc70, BAG2, hnRNP M, and ubiquitin proteins, known to be components of different cellular protein machines, were performed by LC-MS/MS (Figure 6). The patterns were compared with the size profiles obtained by gel filtration on Sepharose 6 of extracts of HEK293 cells transfected with the EBNA5 expression vector pCI-4(W1W2)Y1Y2 or the empty pCI vector. The eluates were analyzed by immunoblotting for EBNA5 and the endogenously expressed proteins Hsp70, BAG2, and CHIP (Figure.7). The results obtained with the EBNA5-StrepTAP protein complex purification procedure showed that the major fraction of EBNA5 in the cells eluted in the void volume. The results indicated that EBNA5 is part of high molecular weight protein complexes of 2000 kDa or more. The elution profiles of whole 2316

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Forsman et al.

Figure 6. Elution profile from Superose 6 gel filtration of StrepTAP eluates. The protein elution profile was measured by UV absorbance at 280 nm. The elution behavior of a protein standard with defined molecular masses is indicated at the top. The presence of EBNA5, Hsp70, Hsc70, BAG2, hnRNP M and Ubiquitin in fractions collected during gel filtration was verified by LC-MS/MS and plotted in the lower panel of the figure (p, present; -, not detected). As seen in the diagram, EBNA5 and the interacting protein complexes eluted mainly with the void volume, indicating multiprotein complex sizes of over 2000 kDa. A smaller protein complex involving Hsp70 and Hsc70 was also observed at approximately 200 kDa.

cell lysates with regard to endogenously expressed Hsp70, BAG2, and CHIP proteins showed that the individual proteins fractionated in complexes with sizes ranging from 200 to >2000 kDa (Figure 7). The size distribution of endogenous Hsp70, BAG2 and CHIP containing protein complexes in mock transfected cells were approximately the same as those reported by others.39 We concluded that our results are consistent with the notion that the TAP tag method allows purification of delicate subcellular structures of complex nature. They also provide support to the idea that the functional units of the cell can form interconnected structures.

Discussion The mechanism through which EBNA5 functions as a coactivator of EBV-induced B-cell immortalization remains an important question in EBV biology. EBNA5 has been shown to interact with a considerable number of cellular proteins, but in many cases, without clarification of the functional aspects of the interaction, although evidence consistent with the notion that EBNA5 is a protein with several functions has accumulated. To further elucidate the functions, we resorted to the strategy of identifying the interaction partners of EBNA5 in the absence of EBNA2. To that end, we employed an efficient tandem affinity purification protocol that allowed protein complex purification under native conditions from mammalian cells. We isolated EBNA5-cell protein complexes, analyzed and identified the protein components by LC-MS/MS followed by searches against a protein sequence database. To minimize the occurrence of false positive protein identifications, stringent criteria were set in the handling of database search results. To get an estimate of the rate of false positive protein identifications, searches were performed against a randomized database. The procedure employed showed to be a robust general technique that enables efficient and systematic purification of protein complexes from mammalian cells. The technique can be used

EBNA5 Multi Protein Complexes

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Figure 7. Gel filtration analysis of multiprotein complexes in whole cell lysates. Whole cell lysates from pCI-EBNA5 or mock transfected HEK293 cells were size fractionated by Superose 6 chromatography. Presence of specific proteins in the eluted fractions was analyzed by Western blot. The elution behavior of a protein standard with defined molecular masses is indicated at the top. The results show that by this method no differences in protein complex composition can be detected when comparing EBNA5 transfected-, pCI transfected(data not shown) and mock transfected HEK 293 cells. The distribution of the protein components was similar as reported by others.39

with different bait proteins. The obvious risk that proteins with low abundance or low complex binding affinity are not identified still has to be considered. A total of 147 novel putative EBNA5 interaction partners were identified. Of the 37 validated interactors, the majority belonged to one of three functional groups: pre-mRNA processing, protein folding and degradation and ribosomal proteins (Table 1). Consistent with published data, Hsp70, Hsc70, HA95, prolyl-4-hydroxylase, Hsp27, and R- and β-tubulin were shown to interact with EBNA5. Our study did not identify the pRb, p53, hERR-1, DNA-PKcs or Sp100 proteins as EBNA5 associated partners, although such interactions have been established with other techniques by other groups.13–15,19,22,25 There could be many different explanations for this difference, one of them exemplified by the known fact that HEK293 cells contain only very low levels of Sp100. Our finding that the Bcl2-associated Athanogene 2 (BAG2) and the 26S Proteasome non-ATPase subunit 2 are members of the EBNA5 interactor protein family, as is the previously wellcharacterized heat shock protein Hsp70, might have interesting implications with regard to EBNA5 functions. These proteins constitute components of the cytoplasmic protein quality control systems that recognize proteins with abnormal structures and either refold them to normal conformation or target them for degradation. The chaperone-dependent protein degradation predominantly occurs through the ubiquitin-proteasome pathway. The 26S proteasome is the major intracellular protease in eukaryotes and the only protease known to degrade polyubiquitinated proteins. Notably, our list of identified and verified EBNA5 interacting partners included the non-ATPase subunit 2 of the 26S proteasome. Hsp70 belongs to the protein family of molecular chaperones characterized by the ability to associate with non-native proteins. The members are generally considered as cellular folding and assembly factors. In recent years, it has become apparent that they also play an important role in degradation of misfolded proteins. Hsp70 and other chaperones cooperate with co-chaperones that regulate the ATPase cycle, assist substrate loading onto the chaperones, and recruit the chaperones to diverse protein complexes and subcellular locations.

The engagement of Hsp70 in a specific cellular process, thus, depends on its cooperation with a distinct set of co-chaperones. Labeling chaperone substrates for degradation depends on the activity of chaperone-associated ubiquitin ligases. The key component in this activity is the C terminus of Hsp70interacting protein (CHIP). CHIP and its partner Ubc (ubiquitin-conjugating enzyme) mediate the ubiquitination of chaperone substrates that are presented by Hsp70, thereby initiating sorting of proteins to the proteasome and degradation. The fact that chaperone-assisted degradation proceeds normally also in cells that lack CHIP demonstrates the existence of other ubiquitin ligases that can cooperate with Hsp70 and Hsp90. Co-chaperones, including the EBNA5 interactor BAG2 identified here, not only facilitate chaperone-assisted degradation, but also control and restrict degradation functions of molecular chaperones. BAG2 binds to the Hsp70/CHIP complex through recognition of the ATPase domain of Hsp70 and inhibits CHIPmediated ubiquitination by abrogation of the interaction between CHIP and its partner ubiquitin conjugating enzyme.39,40 BAG2 utilizes the ATPase domain of Hsp70 as the docking site and, thus, competes with the degradation stimulating cochaperone BAG1 in the regulation of the Hsp70/CHIP complex. Thus, preventing CHIP binding to Hsp70 seems to be another strategy to control the degradation-inducing activity of the ubiquitin ligase. The concerted action of competing and cooperating co-chaperones, thus, can give rise to functionally distinct chaperone machines. We suggest as a working hypothesis that the EBNA5/Hsp70/ BAG2 complex identified in the present study represent a chaperone complex that has been turned into a machine with protein folding activity and abrogated protein degradation ability by the presence of EBNA5. Notably, CHIP is absent from the isolated complex. We have confirmed this observation by independent experiments both in HEK293 cells with our standard two-step TAP tag procedure and by co-immunoprecipitation in lymphoid cells (data not shown). Thus, the concerted action of BAG2 and EBNA5 might repress and displace CHIP from Hsp70 in the chaperone complex and tip the functional balance from ubiquitin-proteasome mediated Journal of Proteome Research • Vol. 7, No. 6, 2008 2317

research articles degradation of proteins in the direction of increased folding and repair. This might be important particularly during the early phase of primary B-lymphocyte EBV infection where EBNA5 is known to play a major role. Notably, the non-ATPase subunit 2 of 26S proteasome is also an EBNA5 interacting protein. This would be compatible also with a direct inhibitory effect of EBNA5 on ubiquitin-proteasome mediated protein degradation. CHIP and EBNA5 can both interact with Hsp70. Recently, Peng et al.41 reported that overexpression of CHIP strongly down-regulated EBNA5 co-activation. CHIP effects were Hsp70-dependent, indicating a background downmodulating role for CHIP in Hsp70 augmentation of EBNA2 and EBNA5 co-activation. Evidence for a direct interaction between EBNA5 and CHIP was, however, not presented. In the context of inhibition of proteasome activity, it might be worth noting that another virus protein, the adenovirus early region 1A protein (AdE1A), interacts with the S2 subunit of the 19S regulatory complex of the 26S proteasome both in vivo and in vitro.42 Although AdE1A is primarily able to modulate the level of expression of cellular proteins through regulation of transcription, it can also affect the rate of protein degradation by targeting the 26S proteasome. The interaction of AdE1A with the S2 subunit was shown to inhibit the ability of the 26S proteasome to degrade p53 both in vitro and in vivo, suggesting that regulation of p53 level by AdE1A is largely due to direct inhibition of proteasome activity.42

Acknowledgment. We thank Dr. S. Hattori for the pEFBOS/HA-BAG2 plasmid, Dr. M. Swanson for the pHCM4 plasmid and Dr. J. Ho¨hfeld for the BAG2 antibody. The work was funded by the Swedish Medical Research Council (project 5667), the Swedish Cancer Society, IngaBritt and Arne Lundberg Foundation, the Assar Gabrielsson Foundation for Clinical Research, the King Gustav V Jubilee Clinic Cancer Research Foundation, and the Sahlgrenska University Hospital. Supporting Information Available: All supplementary tables are listed in an excel file. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Crawford, D. H. Biology and disease associations of Epstein-Barr virus. Philos. Trans. R. Soc. London, Ser. B 2001, 356 (1408), 461– 73. (2) Rickinson, A.; Kieff, E. Epstein-Barr Virus in Fields’ Fundamental Virology, 4th ed.; Lippincott Raven Publishers: Philadephia, PA, 2001; pp 2575-627. (3) Bornkamm, G. W.; Hammerschmidt, W. Molecular virology of Epstein-Barr virus. Philos. Trans. R. Soc. London, Ser. B 2001, 356 (1408), 437–59. (4) Ansieau, S.; Scheffrahn, I.; Mosialos, G.; Brand, H.; Duyster, J.; Kaye, K.; Harada, J.; Dougall, B.; Hubinger, G.; Kieff, E.; Herrmann, F.; Leutz, A.; Gruss, H. J. Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation [retracted by Kieff, E. Proc. Natl. Acad. Sci. U.S.A. 1997, 94 (23), 12732] Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (24), 14053-8. (5) Harada, S.; Kieff, E. Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. J. Virol. 1997, 71 (9), 6611–8. (6) Masciarelli, S.; Mattioli, B.; Galletti, R.; Samoggia, P.; Chichiarelli, S.; Mearini, G.; Mattia, E. Antisense to Epstein Barr Virus-encoded LMP1 does not affect the transcription of viral and cellular proliferation-related genes, but induces phenotypic effects on EBVtransformed B lymphocytes. Oncogene 2002, 21 (26), 4166–70. (7) McCann, E. M.; Kelly, G. L.; Rickinson, A. B.; Bell, A. I. Genetic analysis of the Epstein-Barr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function. J. Gen. Virol. 2001, 82 (Pt. 12), 3067–79.

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