Protein Profiling with Epstein-Barr Nuclear Antigen-1 Reveals an Interaction with the Herpesvirus-associated Ubiquitin-specific Protease HAUSP/USP7
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m303977200
ISSN1083-351X
AutoresMelissa N. Holowaty, Mahel Zeghouf, Hong Wu, Judy Tellam, Vicki Athanasopoulos, Jack Greenblatt, Lori Frappier,
Tópico(s)Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
ResumoThe Epstein-Barr nuclear antigen-1 (EBNA1) protein of Epstein-Barr virus is important for the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes; has been implicated in host cell immortalization; and avoids proteasomal processing and cell-surface presentation. To gain insight into how EBNA1 fulfills these functions, we have profiled cellular protein interactions with EBNA1 using EBNA1 affinity chromatography and tandem affinity purification (TAP) of EBNA1 complexes from human cells (TAP-tagging). We discovered several new specific cellular protein interactions with EBNA1, including interactions with HAUSP/USP7, NAP1, template-activating factor-Iβ/SET, CK2, and PRMT5, all of which play important cell regulatory roles. The ubiquitin-specific protease USP7 is a known target of herpes simplex virus, and the USP7-binding region of EBNA1 was mapped to amino acids 395–450. A mutation in EBNA1 that selectively disrupted binding to USP7 was found to cause a 4-fold increase in EBNA1 replication activity but had no effect on EBNA1 turnover and cell-surface presentation. The results suggest that USP7 can regulate the replication function of EBNA1 and that EBNA1 may influence cellular events by sequestering key regulatory proteins. The Epstein-Barr nuclear antigen-1 (EBNA1) protein of Epstein-Barr virus is important for the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes; has been implicated in host cell immortalization; and avoids proteasomal processing and cell-surface presentation. To gain insight into how EBNA1 fulfills these functions, we have profiled cellular protein interactions with EBNA1 using EBNA1 affinity chromatography and tandem affinity purification (TAP) of EBNA1 complexes from human cells (TAP-tagging). We discovered several new specific cellular protein interactions with EBNA1, including interactions with HAUSP/USP7, NAP1, template-activating factor-Iβ/SET, CK2, and PRMT5, all of which play important cell regulatory roles. The ubiquitin-specific protease USP7 is a known target of herpes simplex virus, and the USP7-binding region of EBNA1 was mapped to amino acids 395–450. A mutation in EBNA1 that selectively disrupted binding to USP7 was found to cause a 4-fold increase in EBNA1 replication activity but had no effect on EBNA1 turnover and cell-surface presentation. The results suggest that USP7 can regulate the replication function of EBNA1 and that EBNA1 may influence cellular events by sequestering key regulatory proteins. Epstein-Barr virus (EBV) 1The abbreviations used are: EBV, Epstein-Barr virus; EBNA1, Epstein-Barr nuclear antigen-1; TAP, tandem affinity purification; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TAF, template-activating factor; TBP, TATA box-binding protein.1The abbreviations used are: EBV, Epstein-Barr virus; EBNA1, Epstein-Barr nuclear antigen-1; TAP, tandem affinity purification; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TAF, template-activating factor; TBP, TATA box-binding protein. is a ubiquitous human γ-herpesvirus that persists for the life of the host. As part of its latent infectious cycle, EBV immortalizes the host cell and, in doing so, predisposes the cell to malignant transformation. As a result, EBV is associated with several types of cancer. EBV genomes are maintained in latently infected replicating cells as circular DNA episomes that replicate once per cell cycle and segregate stably during cell division (reviewed in Refs. 1Kieff E. Rickinson A.B. Knipe D.M. Howley P.M. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2001: 2511-2573Google Scholar and 2Frappier L. Funnell B.E. Phillips G. The Biology of Plasmids. American Society for Microbiology, Washington, D. C.2003Google Scholar). Epstein-Barr nuclear antigen-1 (EBNA1) is the only viral protein required to maintain the EBV genomes in proliferating cells, which it does by binding to recognition sites in the FR (family of repeats) and DS (dyad symmetry) elements of the latent origin of DNA replication, oriP (3Yates J.L. Warren N. Sugden B. Nature. 1985; 313: 812-815Crossref PubMed Scopus (979) Google Scholar, 4Reisman D. Yates J. Sugden B. Mol. Cell. Biol. 1985; 5: 1822-1832Crossref PubMed Scopus (312) Google Scholar). EBNA1 binding to the DS element is necessary to initiate DNA replication from this element (5Harrison S. Fisenne K. Hearing J. J. Virol. 1994; 68: 1913-1925Crossref PubMed Google Scholar). EBNA1 binding to the FR element is important for the partitioning of the EBV episomes during cell division and also activates the expression of other viral latency genes (6Reisman D. Sugden B. Mol. Cell. Biol. 1986; 6: 3838-3846Crossref PubMed Scopus (252) Google Scholar). In addition to its functions at oriP, EBNA1 has been shown to repress its own transcription (7Sample J. Henson E.B. Sample C. J. Virol. 1992; 66: 4654-4661Crossref PubMed Google Scholar) and to promote the development of B-cell lymphomas in transgenic mice, suggesting a direct role for EBNA1 in cell transformation (8Wilson J. Bell J. Levine A. EMBO J. 1996; 15: 3117-3126Crossref PubMed Scopus (276) Google Scholar).While fulfilling all of its functions, EBNA1 avoids detection by host cytotoxic T-lymphocytes. This ability to hide from the immune system is biologically important, as it enables the persistence of latently infected cells that express EBNA1 in the absence of other EBV antigens. The failure of EBNA1 to elicit a cytotoxic T-lymphocyte response is due to lack of proteasomal processing, which prevents the presentation of EBNA1 by major histocompatibility complex class I molecules on the cell surface (9Levitskaya J. Coram M. Levitsky V. Imreh S. Steigerwald-Mullen P.M. Klein G. Kurilla M.G. Masucci M.G. Nature. 1995; 375: 685-688Crossref PubMed Scopus (664) Google Scholar). This property of EBNA1 has been attributed to the central Gly-Ala repeat, which varies in length in different EBV isolates and is not required for any of the EBNA1 functions measured in tissue culture (10Levitskaya J. Sharipo A. Leonchiks A. Ciechanover A. Masucci M.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12616-12621Crossref PubMed Scopus (431) Google Scholar, 11Blake N. Lee S. Redchenko I. Thomas W. Steven N. Leese A. Steigerwald-Mullen P. Kurilla M.G. Frappier L. Rickinson A. Immunity. 1997; 7: 791-802Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar).EBNA1 has no apparent enzymatic activities and is thought to fulfill its functions by mediating interactions with specific host cellular proteins. However, few of these cellular protein interactions have been identified. To date, only yeast one- and two-hybrid approaches have been used to screen for EBNA1-interacting proteins; these screens have identified importin-α (also called karyopherin-α2 or Rch1), karyopherin-α1, p32/TAP, and EBP2 as EBNA1-binding proteins (12Kim A.L. Maher M. Hayman J.B. Ozer J. Zerby D. Yates J.L. Lieberman P.M. Virology. 1997; 239: 340-351Crossref PubMed Scopus (37) Google Scholar, 13Wang Y. Finan J.E. Middeldorp J.M. Hayward S.D. Virology. 1997; 236: 18-29Crossref PubMed Scopus (136) Google Scholar, 14Fischer N. Kremmer E. Lautscham G. Mueller-Lantzsch N. Grasser F.A. J. Biol. Chem. 1997; 272: 3999-4005Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar, 16Ito S. Ikeda M. Kato N. Matsumoto A. Ishikawa Y. Kumakubo S. Yanagi K. Virology. 2000; 266: 110-119Crossref PubMed Scopus (29) Google Scholar, 17Aiyar A. Tyree C. Sugden B. EMBO J. 1998; 17: 6394-6403Crossref PubMed Scopus (109) Google Scholar). Since importin-α and karyopherin-α1 are known nuclear transport factors, their interaction with EBNA1 may be important for EBNA1 entry into the nucleus. p32/TAP (also called gC1q-R) is predominantly a mitochondrial protein, but has been found to bind to a wide variety of proteins with diverse functions (Refs. 13Wang Y. Finan J.E. Middeldorp J.M. Hayward S.D. Virology. 1997; 236: 18-29Crossref PubMed Scopus (136) Google Scholar and 18Muta T. Kang D. Kitajima S. Fujiwara T. Hamasaki N. J. Biol. Chem. 1997; 272: 24363-24370Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 19Luo Y. Yu H. Peterlin B.M. J. Virol. 1994; 68: 3850-3856Crossref PubMed Google Scholar, 20Braun L. Ghebrehiwet B. Cossart P. EMBO J. 2000; 19: 1458-1466Crossref PubMed Scopus (249) Google Scholar and references therein). Thus, the significance of the interaction of EBNA1 with p32/TAP remains unclear. The interaction of EBNA1 with EBP2 seems to be important for the segregation function of EBNA1. EBP2 is a component of the cellular mitotic chromosomes, and EBNA1 appears to attach to EBP2 to partition EBV plasmids (15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar, 21Wu H. Ceccarelli D.F. Frappier L. EMBO Rep. 2000; 1: 140-144Crossref PubMed Scopus (85) Google Scholar, 22Kapoor P. Shire K. Frappier L. EMBO J. 2001; 20: 222-230Crossref PubMed Scopus (55) Google Scholar). In addition to the EBNA1-binding proteins identified by screening, studies that have specifically tested for interactions of EBNA1 with replication protein A and the origin recognition complex have detected these interactions, suggesting that they might be important for the replication function of EBNA1 (23Zhang D. Frappier L. Gibbs E. Hurwitz J. O'Donnell M. Nucleic Acids Res. 1998; 26: 631-637Crossref PubMed Scopus (46) Google Scholar, 24Dhar S.K. Yoshida K. Machida Y. Khaira P. Chaudhuri B. Wohlschlegel J.A. Leffak M. Yates J. Dutta A. Cell. 2001; 106: 287-296Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar).Since the present set of known protein interactions with EBNA1 is unlikely to account for all of the EBNA1 functions, we have used additional methods to screen for human proteins that specifically recognize EBNA1. Using affinity chromatography and tandem affinity purification (TAP)-tagging approaches, we have identified novel EBNA1-interacting proteins, including the deubiquitinating enzyme USP7 (also known as HAUSP).EXPERIMENTAL PROCEDURESPurification of EBNA1 and Δ325–376 —EBNA1 (lacking most of the Gly-Ala repeat region) was expressed as a hexahistidine fusion from a baculovirus. EBNA1 was purified from insect cell nuclei on a metal chelating column, followed by a heparin-agarose column (25Frappier L. O'Donnell M. J. Biol. Chem. 1991; 266: 7819-7826Abstract Full Text PDF PubMed Google Scholar). Δ325–376 (lacking amino acids 325–376 in addition to most of the Gly-Ala repeat) was expressed as a hexahistidine fusion from pET15b in Escherichia coli and purified as described for EBNA1.EBNA1 Affinity Columns—HeLa S3 cells (10 g; National Cell Culture Center) were lysed in 11 ml of 10 mm HEPES (pH 7.5), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol (DTT), and complete protease inhibitors (Roche Applied Science). 10 ml of 50 mm HEPES (pH 7.5), 1.5 mm MgCl2, 1.26 m NaCl, 0.5 mm DTT, 0.6 mm EDTA, and 75% glycerol was added, and the lysate was homogenized in a Dounce homogenizer. After 30 min on ice, the extract was clarified by centrifugation for3hat 64,000 × g and then dialyzed overnight against 50 mm HEPES (pH 7.5), 20% glycerol, 0.5 mm DTT, 5 mm MgCl2, and 75 mm KCl. Prior to loading onto affinity columns, CaCl2 was added to 4 mm, and lysates were incubated with RNase A and DNase I (1.4 μg/mg of lysate) for 30 min at 25 °C to remove nucleic acid.Purified EBNA1 or Δ325–376 was covalently linked to Affi-Gel 10 (Bio-Rad) by incubating 2 ml of protein/ml of Affi-Gel in buffer A (20 mm HEPES (pH 7.5), 10% glycerol, 0.1 mm EDTA, 1 mm DTT, and 1 m NaCl) and blocked as described previously (26Sopta M. Carthew R.W. Greenblatt J. J. Biol. Chem. 1985; 260: 10353-10360Abstract Full Text PDF PubMed Google Scholar). Coupled resin was washed with buffer A and equilibrated in buffer A containing 0.1 m NaCl (buffer B) before pouring into 40-μl microcolumns. 400 μl of HeLa lysate (at 14 mg/ml) was applied to the columns. Columns were washed with 400 μl of buffer B and 160 μl of buffer B containing 1% Triton X-100 and then sequentially eluted with buffer A and 1% SDS. For rechromatography assays, the buffer A eluates from three columns were pooled, dialyzed against buffer A containing 200 mm NaCl, and reapplied to an EBNA1 affinity column. This column was washed and eluted as described above. Column eluates were analyzed by SDS-PAGE and silver staining.Mass Spectrometry—Gel slices containing the protein bands were identified by reduction in DTT, alkylation in iodoacetamide, and then subjection to in-gel trypsin hydrolysis. The peptides were purified and analyzed by MALDI-TOF mass spectrometry using a cyano-4-hydroxycinnamic acid matrix (Sigma) on a Voyager DE-STR instrument (Applied Biosystems) (27Mann M. Hojrup P. Roepstorff P. Biol. Mass Spectrom. 1993; 22: 338-345Crossref PubMed Scopus (831) Google Scholar). Identification of the proteins using these mass fingerprinting data was carried out using the ProFound software. 2Available at 129.85.19.192/profound_bin/WebProFound.exe.Co-immunoprecipitation from Insect Cells—Baculoviruses expressing EBNA1GA (with a long Gly-Ala repeat) (11Blake N. Lee S. Redchenko I. Thomas W. Steven N. Leese A. Steigerwald-Mullen P. Kurilla M.G. Frappier L. Rickinson A. Immunity. 1997; 7: 791-802Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), EBNA1 or EBNA1 mutants 330–641, 330–619, 452–641, and Δ41–376 have all been described previously (28Goldsmith K. Bendell L. Frappier L. J. Virol. 1993; 67: 3418-3426Crossref PubMed Google Scholar, 29Avolio-Hunter T.M. Frappier L. Nucleic Acids Res. 1998; 26: 4462-4470Crossref PubMed Scopus (24) Google Scholar). The baculovirus expressing EBNA1 with a small Gly-Ala repeat and lacking amino acids 395–450 was constructed by QuikChange mutagenesis (Stratagene) of EBNA1 in pFast-Bac (Invitrogen). Baculovirus was generated according to the manufacturer's specifications. A baculovirus expressing USP7 with an N-terminal hexahistidine tag was similarly constructed using USP7 cDNA in pET-3a, kindly provided by Roger Everett (30Everett R. Meredith M. Orr A. Cross A. Kathoria M. Parkinson J. EMBO J. 1997; 16: 1519-1530Crossref PubMed Scopus (315) Google Scholar). For co-immunoprecipitation assays, 107 Sf9 cells were infected with baculovirus expressing USP7 and/or EBNA1 or an EBNA1 mutant. 32 h post-infection, cells were labeled with 50 μCi of [35S]methionine for 16 h as described previously (15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar). Cells were lysed in 1 ml of immunoprecipitation buffer (20 mm Tris-HCl (pH 7.5), 200 mm NaCl, 1 mm MgCl2, 10% glycerol, 1% Triton X-100, and protease inhibitors) on ice for 30 min. After centrifugation, the supernatant was precleared with protein A-Sepharose and then precipitated with rabbit anti-EBNA1 polyclonal antibody K67 (kindly provided by Jaap Middeldorp) and protein A-Sepharose as described previously (15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar). Immunoprecipitated proteins were analyzed by SDS-PAGE, followed by autoradiography of the dried gels.TAP-tagging Experiments—The DNAs coding for EBNA1, Δ395–450, and Δ325–376 were PCR-amplified from plasmids containing these EBNA1 mutants and cloned between the XhoI and NotI sites of pMZI. pMZI expresses proteins with C-terminal TAP tags (31Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Seraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2275) Google Scholar) in mammalian cells under the control of an ecdysone-inducible promoter. The pMZI vector and the pMZI-LacZ construct, which encodes TAP-tagged β-galactosidase, will be described elsewhere. 3M. Zeghouf, J. Li, G. Butland, A. Borkowska, V. Canadien, D. Richards, B. Beattie, A. Emili, and J. Greenblatt, submitted for publication. 293T cells at 60% confluence in 150-mm dishes were cotransfected by calcium phosphate precipitation with 8 μg of pMZI expressing EBNA1 or an EBNA1 mutant and 8 μg of pVgRxR (Invitrogen), which encodes the ecdysone receptor heterodimer. The precipitate was removed 15 h post-transfection, and expression of the EBNA1 proteins was induced by adding medium containing 3 μm Ponasterone A (Invitrogen). The cells were harvested 22–48 h later, and a whole cell extract was prepared from 4 × 107 cells as described by Xiao et al. (32Xiao H. Friesen J.D. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar), except that the extract was dialyzed against 10 mm HEPES (pH 7.9), 0.1 m potassium acetate, 0.1 mm EDTA, 0.1 mm DTT, and 10% glycerol. The extract was mixed with 50 μl of IgG-Sepharose 6 beads (Amersham Biosciences) for 3 h at 4 °C, and bound TAP-tagged protein was released by incubation overnight with 30 units of tobacco etch virus (TEV) protease (Invitrogen) in 10 mm Tris-HCl (pH 8), 100 mm NaCl, 0.1% Triton X-100, 0.5 mm EDTA, 1 mm DTT, and 10% glycerol. Eluted protein was further purified on a 40-μl column of calmodulin-Sepharose 4B. Protein complexes were eluted with 10 mm Tris-HCl (pH 8), 100 mm NaCl, 2 mm EGTA, 10 mm β-mercaptoethanol, 1 mm imidazole, and 0.1% Triton X-100; concentrated by lyophilization; and analyzed by SDS-PAGE and silver staining. Protein bands were identified by MALDI-TOF mass spectrometry.EBNA1 Functional Assays—The construction of pc3oriPEBNA1 and pc3oriP, which contain the EBV oriP sequence and express EBNA1 or no protein, respectively, has been described previously (15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar). Plasmid pc3oriPΔ395–450 was constructed by QuikChange mutagenesis of pc3oriPEBNA1. Transient replication, plasmid maintenance, and transcriptional activation assays were performed as described previously (33Wu H. Kapoor P. Frappier L. J. Virol. 2002; 76: 2480-2490Crossref PubMed Scopus (102) Google Scholar). For replication assays, C33A cells were transfected with 10 μg of pc3oriP plasmids; and 72 h post-transfection, plasmids were recovered, linearized, digested with DpnI, and analyzed by Southern blotting. For plasmid maintenance assays, C33A cells were transfected with 1 μg of plasmid; and after 14 days of selection in G418, plasmids were linearized, digested with DpnI, and analyzed by Southern blotting. Plasmid bands were visualized by autoradiography and quantified by PhosphorImager analysis using ImageQuant software (Amersham Biosciences). For transactivation assays, C33A cells were transfected with 5 μg of pc3oriP plasmids and 2 μg of the pFRTKCAT reporter construct (kindly provided by Bill Sugden). 24 h later, cell lysates were prepared, and 50 μg of each was assayed for chloramphenicol acetyltransferase activity using several reaction times as described previously (34Ceccarelli D.F. Frappier L. J. Virol. 2000; 74: 4939-4948Crossref PubMed Scopus (61) Google Scholar). Reaction products at each time point were quantified by PhosphorImager analysis and used to determine the acetylation rate.Protein Turnover Assays—2 × 106 293T cells were transfected with 5 μg of pc3oriPEBNA1 or pc3oriPΔ395–450 using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's recommendations. Cells were grown for 1 week under selection for the plasmid (400 μg/ml G418) before blocking the cells in 100 μg/ml cycloheximide (Sigma). Cells were harvested at various times after blocking by resuspending in 9 m urea and 5 mm Tris-HCl (pH 7.5). Lysates were clarified by sonication and centrifugation, and 50 μg of each was separated by SDS-PAGE and Western-blotted using anti-EBNA1 antiserum R4 (raised against EBNA1-(452–641)). The same lysates were also probed with anti-interferon regulatory factor 1 (IRF-1) antibody (C20, Santa Cruz Biotechnology).Immunofluorescence Microscopy—C33A cells expressing EBNA1 were generated by transfecting C33A cells with pc3oriPEBNA1 and growing the cells under selection for the plasmid. The cells were then grown on coverslips to 60% confluence, fixed with formaldehyde (5% (v/v) in phosphate-buffered saline containing 2% sucrose), and permeabilized with acetone/methanol (70:30) at –20 °C. Coverslips were washed with phosphate-buffered saline and blocked for 1 h in 10% bovine serum albumin. The cells were stained with anti-EBNA1 monoclonal antibody OT1x (kindly supplied by Jaap Middeldorp) at a 1:150 dilution and rabbit anti-USP7 antibody r201 (kindly provided by Roger Everett) at a 1:200 dilution, followed by staining with Texas Red-labeled goat anti-mouse (Molecular Probes, Inc.) and fluorescein isothiocyanate-labeled goat anti-rabbit (Invitrogen) secondary antibodies (at 1:200 and 1:30 dilutions, respectively). Cells were then counterstained with 4,6-diamidino-2-phenylindole. The slides were mounted in 5 μl of antifade solution and observed at a magnification of ×400 using a Leica DMR microscope and Openlab software. For microscopy of Raji cells, cells were allowed to adhere to coverslips coated with poly-l-lysine before fixing with paraformaldehyde (3% in phosphate-buffered saline), permeabilizing in 1% Triton X-100 and phosphate-buffered saline, and blocking as described above. Staining and microscopy were performed as described for C33A cells, except that rabbit anti-USP7 antiserum r2b2, raised against full-length USP7, was used (at a dilution of 1:200).RESULTSEBNA1-interacting Proteins Identified by Affinity Chromatography—EBNA1-binding proteins were isolated from a HeLa cell extract by retention on columns containing purified EBNA1. Elution profiles were compared for EBNA1 columns and negative control columns (with no protein coupled) in four separate experiments, and typical results are shown in Fig. 1A. Proteins found to be specifically and reproducibly retained on the EBNA1 columns were identified by MALDI-TOF mass spectrometry. Two of these proteins, importin-α and p32/TAP, were previously identified as EBNA1-interacting proteins in two-hybrid screens (12Kim A.L. Maher M. Hayman J.B. Ozer J. Zerby D. Yates J.L. Lieberman P.M. Virology. 1997; 239: 340-351Crossref PubMed Scopus (37) Google Scholar, 13Wang Y. Finan J.E. Middeldorp J.M. Hayward S.D. Virology. 1997; 236: 18-29Crossref PubMed Scopus (136) Google Scholar, 14Fischer N. Kremmer E. Lautscham G. Mueller-Lantzsch N. Grasser F.A. J. Biol. Chem. 1997; 272: 3999-4005Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar). In addition, previously unknown interactions were detected with USP7, karyopherin-β3, karyopherin-β2 (not obvious in Fig. 1A, but shown in Figs. 1B and 2), importin-β, NAP1 (nucleosome assembly protein-1), template-activating factor (TAF)-Iα, TAF-Iβ, CK2α (casein kinase-2α), CK2α′, and pp32. USP7 is a ubiquitin-specific protease that has been found to interact with the ICP0 protein of herpes simplex virus type 1 (30Everett R. Meredith M. Orr A. Cross A. Kathoria M. Parkinson J. EMBO J. 1997; 16: 1519-1530Crossref PubMed Scopus (315) Google Scholar). Karyopherin-β2 and karyopherin-β3 are nuclear import factors (35Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar). NAP1 is a histone chaperone that facilitates the proper spacing of nucleosomes (36Ishimi Y. Kojima M. Yamada M. Hanaoka F. Eur. J. Biochem. 1987; 162: 19-24Crossref PubMed Scopus (65) Google Scholar). TAF-Iα and TAF-Iβ form the template-activating factor, which can activate replication and transcription from chromatin templates through histone interactions (37Okuwaki M. Nagata K. J. Biol. Chem. 1998; 273: 34511-34518Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In addition, TAF-Iβ, also known as SET, has been found to regulate p21Cip1 (38Estanyol J.M. Jaumot M. Casanovas O. Rodriguez-Vilarrupla A. Agell N. Bachs O. J. Biol. Chem. 1999; 274: 33161-33165Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). CK2α and CK2α′ are two of the three subunits of the CK2 serine/threonine kinase (39Litchfield D.W. Biochem. J. 2002; 369: 1-15Crossref Google Scholar). pp32 is an acidic nuclear protein that forms part of the INHAT complex (40Seo S.B. McNamara P. Heo S. Turner A. Lane W.S. Chakravarti D. Cell. 2001; 104: 119-130Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). We noted that EBP2, which was found to bind EBNA1 by other methods (15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar), was not among the proteins retained on the EBNA1 column. However, Western blot analysis of the soluble HeLa lysate fraction applied to the column indicated that EBP2 was not present in this extract, but rather had pelleted with the insoluble cellular material. The EBNA1 affinity column experiment was also performed with a B-cell lysate and resulted in the same panel of EBNA1-binding proteins as was isolated from the HeLa lysate (data not shown).Fig. 2Comparison of EBNA1 and Δ325–376 affinity column profiles. HeLa cell lysate was applied to columns containing EBNA1, Δ325–376, or no protein (None). The retained proteins were eluted and analyzed as described for Fig. 1A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since EBNA1 is a highly basic protein (pI 11), we expected that some cellular proteins containing acidic regions would interact with EBNA1 through nonspecific ionic interactions. To identify these proteins, we compared the cellular proteins eluted from the EBNA1 column with those eluted from a column containing TATA box-binding protein (TBP), which has a pI similar to that EBNA1. The elution profile from the TBP column shows that the EBNA1-binding proteins importin-β, TAFI-α, p32/TAP, and pp32 were also retained on the TBP column, suggesting that they interact nonspecifically with basic proteins (Fig. 1A). We also excised bands from the TBP elution profile that migrated at positions similar to those of NAP1, importin-α, and USP7, but MALDI-TOF analyses of these bands indicated that NAP1, importin-α, and USP7 were not present in the TBP eluate. We conclude that USP7, NAP1, importin-α and TAF-Iβ interact specifically with EBNA1. Karyopherin-β and CK2 bands were not observed on the TBP column; however, since they are close to the limit of detection in the EBNA1 profile, we cannot be certain that they are not present in the TBP profile.We next tested the relative affinity of the EBNA1-binding proteins for EBNA1 by rechromatography of EBNA1 column eluates on a second EBNA1 column. Eluates from three EBNA1 columns were pooled, dialyzed to lower the salt to 100 mm, and then reapplied to another EBNA1 column. When the high salt elution profiles were compared from the first- and second-pass EBNA1 columns, bands corresponding to USP7, importin-α, CK2α, CK2α′, and p32/TAP were all observed to be enriched upon rechromatography relative to the other bands (Fig. 1B), suggesting that these proteins bind EBNA1 with higher affinity than the other proteins. Two bands corresponding to NAP1 were identified in this experiment; the lower band is a proteolytic fragment of the full-length protein.We have previously shown that EBNA1 amino acids 325–376 are essential for both the transcriptional activation and partitioning functions of EBNA1, suggesting that these residues mediate interactions with cellular proteins involved in these processes (15Shire K. Ceccarelli D.F.J. Avolio-Hunter T.M. Frappier L. J. Virol. 1999; 73: 2587-2595Crossref PubMed Google Scholar, 34Ceccarelli D.F. Frappier L. J. Virol. 2000; 74: 4939-4948Crossref PubMed Scopus (61) Google Scholar). To uncover cellular proteins that interact with this functionally important region of EBNA1, we constructed affinity columns using an EBNA1 mutant lacking amino acids 325–376 (Δ325–376) and compared the profile of cellular proteins retained on this column with that on an EBNA1 column (Fig. 2). In each of three experiments, importin-α and NAP1 were not detected on the Δ325–376 column, indicating that residues 325–376 are important for importin-α and NAP1 binding. No other consistent differences were observed between the EBNA1 and Δ325–376 profiles.Mapping of the USP7-interacting Region of EBNA1—The EBNA1-USP7 interaction had not been previously identified and passed all of our criteria for a bona fide interaction, viz. reproducibility, specificity, and strength of binding. To further verify this interaction and to determine whether it is direct or mediated by another human protein, we coexpressed both proteins in insect cells using baculoviruses, metabolically labeled the cells, and performed co-immunoprecipitation experiments using anti-EBNA1 antibody (Fig. 3B). Immunoprecipitates from cell lysates expressing both EBNA1 and USP7 contained labeled bands corresponding to EBNA1 and USP7 (lanes 5 and 14). USP7 also immunoprecipitated with a version of EBNA1 containing a longer Gly-Ala repeat region (EBNA1GA) (lane 13). The USP7 band was not detected in anti-EBNA1 immunoprecipitates from cells expressing either USP7 alone or EBNA1 alone (lanes 8 and 15). Thus, the EBNA1-USP7 interaction does not require other human proteins.Fig. 3Mapping of the USP7-binding region of EBNA1 by co-immunoprecipitation. A, shown is a schematic representation of EBNA1 and EBNA1 mutants and summary of their ability to bind USP7 in the co-immu
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