Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment
2004; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m411405200
ISSN1083-351X
AutoresAyumi Kudoh, Masatoshi Fujita, Lumin Zhang, Noriko Shirata, Tohru Daikoku, Yutaka Sugaya, Hiroki Isomura, Yukihiro Nishiyama, Tatsuya Tsurumi,
Tópico(s)Polyomavirus and related diseases
ResumoWhen exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication. When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication. Eukaryotic cells exhibit a variety of physiological responses, including cell cycle arrest, activation of DNA repair and apoptosis, upon DNA damage. Sets of checkpoint proteins that have been conserved with evolution are rapidly induced to prevent replication or segregation of damaged DNA before repair is completed. The related phosphatidylinositol 3-like kinases, ataxia telangiectasia-mutated (ATM) 1The abbreviations used are: ATM, ataxia telangiectasia-mutated; EBV, Epstein-Barr virus; PBS, phosphate-buffered saline; h.p.i., h post-induction; Gy, Gray; ATR, ATM-Rad3-related; DSB, double-stranded break; FISH, fluorescence in situ hybridization; BrdUrd, bromodeoxyuridine; MRN, Mre11-Rad50-Nbs1; mCSK butter, modified cytoskelton butter; Pipes, 1,4-piperazinediethanesulfonic acid. and ATM-Rad3-related (ATR), respond to a variety of abnormal DNA structures and initiate signaling cascades leading to a DNA damage checkpoint (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). ATM responds to the presence of DNA double-strand breaks (DSBs) induced by ionizing radiation (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2178) Google Scholar). On the other hand, the ATR pathway can be stimulated by hydroxyurea, UV light, and base-damaging agents that interfere with the movement of replication forks (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). The ATR pathway also responds to DSBs, but more slowly than ATM (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2673) Google Scholar). A variety of checkpoint proteins have been identified as substrates for ATM and ATR kinases, including the checkpoint kinases Chk1 and Chk2, as well as γH2AX (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1722) Google Scholar) and p53 (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2178) Google Scholar, 6Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. 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Biol. 2002; 3: 317-327Crossref PubMed Scopus (721) Google Scholar). The MRN complex consisting of Mre11, Rad50, and Nbs1 has been proposed to facilitate ATM activation (15Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (854) Google Scholar, 16Usui T. Ogawa H. Petrini J.H. Mol. Cell. 2001; 7: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 17Williams B.R. Mirzoeva O.K. Morgan W.F. Lin J. Dunnick W. Petrini J.H. Curr. Biol. 2002; 12: 648-653Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and recently demonstrated to function upstream of ATM activation as a damage sensor, in addition to acting as an effector of ATM signaling (15Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (854) Google Scholar, 18Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (424) Google Scholar). ATM/ATR-initiated checkpoint signaling induces p53-dependent and p53-independent responses. The p53-dependent cell cycle checkpoint features p21-mediated inactivation of Cdk2/cyclin E (19el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (8054) Google Scholar, 20Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (729) Google Scholar, 21Dulic V. Kaufmann W.K. Wilson S.J. Tlsty T.D. Lees E. Harper J.W. Elledge S.J. Reed S.I. Cell. 1994; 76: 1013-1023Abstract Full Text PDF PubMed Scopus (1448) Google Scholar), while Chk2 inhibits Cdk2/cyclin E activity by phosphorylation of Cdk2 at Tyr-15, via down-regulation of CDC25A phosphatase (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2673) Google Scholar), in a p53-independent fashion. Among Cdk2-targets, the Rb protein is most important for cell cycle progression and the checkpoint pathways result in its hypophosphorylation, leading to G1 or G2/M cell cycle arrest. The Epstein-Barr virus (EBV) is a human herpes virus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing their continuous proliferation. In the B lymphoblastoid cell lines (LCL) only limited numbers of viral genes are usually expressed and there is no production of virus particles, this being called latent infection. In the latent state, EBV maintains its 170 kbp genome as complete, multiple copies of plasmids that are synthesized only once in each S-phase by the host cell replication machinery, following the rules of chromosome replication (22Yates J.L. Guan N. J. Virol. 1991; 65: 483-488Crossref PubMed Google Scholar). EBV-infected cell lines usually contain a small subpopulation of cells that have switched spontaneously from a latent stage of infection into the lytic cycle. The mechanism of switching is not fully understood, but one of the first detectable changes is expression of the BZLF1 immediate-early gene product. The BZLF1 protein, together with the other immediate-early protein, BRLF1 protein, transactivates viral promoters (23Flemington E.K. Goldfeld A.E. Speck S.H. J. Virol. 1991; 65: 7073-7077Crossref PubMed Google Scholar) and leads to an ordered cascade of viral early and late gene expression. Early gene products include proteins involved in viral DNA replication and DNA metabolism. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: BZLF1, an oriLyt-binding protein; BALF5, a DNA polymerase; BMRF1, a polymerase processivity factor; BALF2, a single-stranded DNA-binding protein; and BBLF4, BSLF1, and BBLF2/3, predicted to be helicase-, primase-, and helicase primase-associated proteins, respectively (24Fixman E.D. Hayward G.S. Hayward S.D. J. Virol. 1995; 69: 2998-3006Crossref PubMed Google Scholar). Viral lytic replication occurs in discrete sites in nuclei, called replication compartments in which viral replication proteins are assembled (25Takagi S. Takada K. Sairenji T. Virology. 1991; 185: 309-315Crossref PubMed Scopus (37) Google Scholar). We have previously demonstrated that induction of the EBV lytic program results in inhibition of replication of cellular DNA as well as explosive replication of viral DNA (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). The levels of p53 and CDK inhibitors remain unchanged throughout the lytic infection, while the amounts of cyclin E/A and the hyperphosphorylated form of Rb increase as lytic infection progresses. The resultant S-phase-like cellular condition is found to be essential for the transcription of viral immediate-early and early genes probably attributed to transcription factors such as E2F-1 and Sp1 expressed during S phase (27Kudoh A. Daikoku T. Sugaya Y. Isomura H. Fujita M. Kiyono T. Nishiyama Y. Tsurumi T. J. Virol. 2004; 78: 104-115Crossref PubMed Scopus (64) Google Scholar). It is of interest to determine whether host cells can monitor EBV lytic replication as DNA damage or abnormal DNA and, if so, how EBV blocks the checkpoint signaling to avoid G1 or G2/M cell cycle arrest and apoptosis. We have previously isolated EBV latently infected Tet-BZLF1/B95-8 cells in which exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). Using this system, we show here for the first time that induction of EBV lytic replication elicits a cellular DNA damage response dependent on ATM. DNA damage sensor MRN complex and phosphorylated ATM are recruited to viral replication compartments, presumably recognizing newly synthesized viral DNAs as abnormal DNA structures. However, the ATM checkpoint signaling was blocked at downstream of p53. Therefore, although EBV lytic replication elicits ATM-dependent DNA damage response, the virus can skillfully block the host response and actively promote an S-phase-like environment advantageous for viral lytic replication. Cells—Tet-BZLF1/B95-8 cells, a marmoset B-cell line latently infected with EBV (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar), and Tet-BZLF1/Akata cells, human EBV-positive Burkitt's lymphoma cells (27Kudoh A. Daikoku T. Sugaya Y. Isomura H. Fujita M. Kiyono T. Nishiyama Y. Tsurumi T. J. Virol. 2004; 78: 104-115Crossref PubMed Scopus (64) Google Scholar), were maintained in RPMI medium supplemented with 1 μg/ml of puromycin, 250 μg/ml of hygromycin B, and 10% tetracycline-free fetal calf serum. To induce lytic EBV replication, a tetracycline derivative, doxycycline, was added to the culture medium at a final concentration of 2 μg/ml. B95-8 cells were cultured in RPMI medium supplemented with 10% fetal calf serum. Antibodies—Primary antibodies were purchased from Cell Signaling (ATM-S1981, Chk1, Chk1-S345, Chk2-T68, p95/NBS1-S343, and p53-S15), Genetex (ATM-2C1), BD Transduction Laboratories (Cdk1, Cdk2, Chk2, Mre11, and Nbs1), Oncogene (p53, p21 and MDM2), Santa Cruz Biotechnology (cyclin A-C19, cyclin B1-GCN1, cyclin E -M20, and E2F1-KH95), Upstate (H2AX-S139), Chemicon (EBV BMRF1-R3) and Argene (EBV BRLF1–8C12). Rabbit polyclonal antibodies to BZLF1, BALF2, BBLF2/3, and BALF5 proteins were prepared as described (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). Highly cross-absorbed secondary reagents for dual-color detection (Alexa-488 and 594) were from Molecular Probes. Protein Preparation—Tet-BZLF1/B95-8, Tet-BZLF1/Akata, and B95-8 cells were harvested at the indicated times post-treatment with doxycycline, washed with phosphate-buffered saline (PBS), and treated with lysis buffer (0.02% SDS, 0.5% Triton X-100, 300 mm NaCl, 20 mm Tris-HCl, pH 7.6, 1 mm EDTA, 1 mm dithiothreitol) for 20 min on ice. Multiple protease inhibitors (Sigma; 25μl/ml), 200 μm sodium vanadate, and 20 mm sodium fluoride were added to the buffer. Samples were centrifuged at 18,000 × g for 10 min at 4 °C, and clarified cell extracts were assayed for protein concentration using a Bio-Rad kit. Immunoblot Analysis—Equal amounts of proteins (20∼50 μg) were loaded into each lane for SDS-10% polyacrylamide (acrylamide, 29.2; bisacrylamide, 0.8) gel electrophoresis (SDS-PAGE). To separate high molecular mass proteins (>180 kDa) or phosphorylated form of Rb proteins, gradient SDS-PAGE or SDS-7.5% PAGE (acrylamide, 72; bisacrylamide, 1), respectively, were applied. The proteins were then processed as described previously (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). Detection of target proteins was with an enhanced chemiluminescence detection system (Amersham Biosciences). Immunofluorescence—Cells were treated with 0.5% Triton X-100-mCSK buffer (10 mm Pipes, pH 6.8, 100 mm NaCl, 300 mm sucrose, 1 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 0.5% Triton X-100) for 10 min on ice, followed by fixation with 70% methanol for 20 min on ice. The fixed cells were washed with PBS and blocked for 20 min in 10% normal goat serum in PBS. Staining with a mouse monoclonal antibody to phosphorylated Ser-1981 of ATM, mouse monoclonal antibodies to NBS1, Mre11, and p53, and a rabbit polyclonal antibody to phosphorylated Ser-15 of p53 was performed overnight at 4 °C in PBS containing 0.5% goat serum. Staining with a rabbit polyclonal antibody to BALF2, a mouse monoclonal antibody to BMRF1 and a rabbit polyclonal antibody to BZLF1 was carried out for 1 h at room temperature. Species-specific secondary antibodies were applied for 1 h at room temperature. Alexa-488 and Alexa-594, highly cross-absorbed secondary reagents, were purchased from Molecular Probes and slides were mounted in Vectashield (Vector Labs) and analyzed by fluorescence confocal microscopy. Confocal fluorescence images were captured and processed using a Radiance2000 Confocal System (Bio-Rad). All the primary antibodies were employed at 1:100 dilutions, and the secondary antibodies at 1:500 dilutions. All washes after antibody incubations were performed with 0.05% Tween-20 in PBS at room temperature. The specificity of the second antibodies and reliability of discrimination with fluorescent microscopy filters were tested. When cells were stained singly for either antigen with inappropriate combinations of first and second antibodies, no fluorescence was observed and also no immunofluorescence was observed with alternate filters. For staining the BrdUrd-incorporated DNA, cells were treated for 10 min with 2 n HCl containing 0.5% Triton X-100 to expose the incorporated BrdUrd residues before blocking. The cells were washed and neutralized with 0.1 m sodium tetraborate (pH 9.0) for 5 min of incubation. For BrdUrd staining, Alexa Fluor 488-conjugated anti-BrdUrd mouse monoclonal antibody was used. Fluorescence in Situ Hybridization (FISH)—EBV BamHI-W fragment was labeled with Chroma Tide Alexa Fluor 594-5-dUTP (Molecular Probes, Inc.) and used for the detection of amplified EBV genomes. At first, immunostaining was performed as described above and then refixed in 4% paraformaldehyde to cross-link bound antibodies. After permeabilizing in 0.2% Triton X-100 (20 min on ice), cells were digested with RNase A, dehydrated in ethanol, air-dried, and immediately covered with the probe mixture containing 50% formamide in 2× SSC containing probe DNA (10 ng/μl), 10% dextran sulfate, salmon sperm DNA (0.1 μg/μl), and yeast tRNA (1 μg/μl). Probe and cells were simultaneously heated at 94 °C for 4 min and incubated overnight at 37 °C. After hybridization, specimens were washed at 37 °C with 50% formamide in 2× SSC (two times for 15 min each) and 2× SSC. Finally, cells were equilibrated in PBS and mounted in vectashield (Vector Laboratories, Inc.). Immunoprecipitation—Cells were lysed in 1 ml of EBC lysis buffer (50 mm Tris-HCl pH 8.0, 120 mm NaCl, 0.5% Nonidet P-40) containing 100 mm sodium fluoride, 2 mm sodium vanadate, and protease inhibitor mixture (Sigma; 25 μl/ml), and then sonicated. The lysates were centrifuged at 18,000 × g for 20 min at 4 °C, and immunoprecipitation was performed using 1 mg of the supernatant and 5 μg of anti-BZLF1 protein-specific IgG-beads or control rabbit IgG beads, with gentle rocking for 1 h at 4 °C. Ternary protein A-antibody-antigen complexes were collected by centrifugation and washed three times with NET-gel buffer (50 mm Tris-HCl pH 8.0, 150 mm NaCl, 0.1% Nonidet P-40, and 1 mm EDTA). The immunoprecipitates were subjected to SDS-10%PAGE followed by immunoblotting analyses. In Vitro Protein Kinase Assay—Cells were washed with ice-cold PBS and then lysed with EBC lysis buffer containing 100 mm sodium fluoride, 2 mm sodium vanadate, and multiple protease inhibitors (Sigma; 25 μl/ml) for 5 min on ice followed by centrifugation at 18,000 × g for 20 min at 4 °C. For kinase assays 200 μg of aliquots of protein extracts were incubated with 1 μg of each antibody in a final volume of 500 μl for 1 h at 4 °C to precipitate cyclin-CDK complexes: C-19 against cyclin A, M-20 against cyclin E and GNS1 against cyclin B. Immune complexes absorbed onto protein A-Sepharose were washed twice with ice-cold NET-gel buffer, and then once with basic kinase buffer (200 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol). The immunoprecipitates were resuspended in 24 μl of kinase buffer (200 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 50 μm ATP containing 5 μCi of [γ-32P]ATP), and kinase reactions were carried out for 60 min at 37 °C with 250 ng of histone H1 (Calbiochem) as substrate. Reactions were stopped by addition of 6 μl of 5× SDS gel loading buffer, and the products were resolved by 12% SDS-PAGE followed by autoradiography. Quantification of Viral DNA Synthesis during Lytic Replication— Tet-BZLF1/B95-8 cells were incubated with 2 μg/ml of doxycycline in the presence or absence of 5 mm caffeine and harvested at the indicated times. Total DNAs were purified from a total of 3.5 × 106 cells and quantified. Dot-blot hybridization was performed, and quantification of the copy numbers of viral genome per cell were determined as described previously (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). Induction of the EBV Lytic Program Elicits a Cellular DNA Damage Response—Lytic replication was induced in Tet-BZLF1/B95-8 cells with doxycycline and cells were harvested at the indicated times. Detailed expression profiles of viral proteins after induction of lytic replication with doxycycline were described previously (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). BZLF1 protein became detectable 4 h post-induction (h.p.i.) (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar) and reached a plateau at 48 h.p.i. (Fig. 1A). The other immediate-early protein, the BRLF1 protein also appeared at 6 h.p.i (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar) with a plateau at 48 h.p.i. (Fig. 1A). Viral early gene products, the BALF2 single-stranded DNA-binding protein, the BBLF2/3 helicase-primase-associated protein, the BALF5 polymerase catalytic protein (data not shown), and the BMRF1 Pol accessory protein (data not shown) appeared after 12 h.p.i. and reached a plateau at 24 h.p.i. The ATM kinase responds primarily to DSBs, and this pathway can act during all phases of the cell cycle. It has been recently proposed that ATM is usually present as an inactive multimer, and this is activated by autophosphorylation at Ser-1981 after DNA strand breaks or changes in the chromatin structure (11Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2750) Google Scholar). As shown in Fig. 1B, immunoblotting of cell lysates revealed that the levels of the phosphorylated form of ATM at Ser-1981 increased upon induction, although the total levels of ATM remained constant throughout lytic infection. This was not the case with B95-8 cells treated with doxycycline. In the presence of DSBs, activated ATM is known to phosphorylate Thr-68 on Chk2, which is required for its activation (9Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (265) Google Scholar, 28Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar). Immunoblotting with anti-Chk2 Thr-68-specific antibody showed phosphorylation of Chk2 at Thr-68 (Fig. 1B), this becoming detectable at 12 h.p.i., reaching a maximum by 24 h.p.i., and then decreasing by degrees. Phosphorylation of histone H2AX, the response of an ATM-controlled, but Chk2-independent branch of ATM signaling (29Yazdi P.T. Wang Y. Zhao S. Patel N. Lee E.Y. Qin J. Genes Dev. 2002; 16: 571-582Crossref PubMed Scopus (412) Google Scholar, 30Falck J. Petrini J.H. Williams B.R. Lukas J. Bartek J. Nat. Genet. 2002; 30: 290-294Crossref PubMed Scopus (322) Google Scholar), was also examined. As shown in Fig. 1B, significant increase was evident at 12 h post-induction. Next, we focused on phosphorylation of p53. Phosphorylation of p53 at Ser-15, a widely accepted target of ATM kinase activity, was conspicuous at 24 h.p.i. However, the EBV lytic replication program had no significant effect on expression levels of p53 protein throughout lytic infection, in agreement with our previous observation (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (87) Google Scholar). It should be noted that not only activated ATM but also ATR kinases phosphorylate Chk2 kinase at Thr-68 and up-regulate its activity (9Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (265) Google Scholar, 28Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar). Phosphorylation of histone H2AX or p53 at Ser-15 is also carried out by ATM/ATR kinases (31Celeste A. Fernandez-Capetillo O. Kruhlak M.J. Pilch D.R. Staudt D.W. Lee A. Bonner R.F. Bonner W.M. Nussenzweig A. Nat. Cell Biol. 2003; 5: 675-679Crossref PubMed Scopus (828) Google Scholar, 32Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (876) Google Scholar). Therefore, EBV lytic replication could activate the ATM, ATR, or both kinases. The ATR kinase responds primarily to DNA replication stress during S phase (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). It can also respond to DSBs if within the S phase, but less efficiently than ATM. In contrast to Chk2, phosphorylation of Chk1 at Ser-345 is known to be carried out mainly by ATR kinase, leading to its activation (10Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (886) Google Scholar). Therefore, we examined Chk1 phosphorylation at Ser-345 in lytic replication-induced B95-8 cells, but no significant phosphorylation was observed (Fig. 1B). Treatment of cells with hydroxyurea, a well-studied activator of the replication checkpoint, clearly induced Chk1 phosphorylation (Fig. 1B), showing the ATR/Chk1 pathway to be intact in the cells. Thus, we conclude that EBV lytic replication elicits activation of ATM DNA damage checkpoint signaling rather than the ATR pathway that responds to replication stress. To ascertain whether lytic replication elicits ATM/Chk2 DNA damage checkpoint signaling in other EBV-latently infected B cells, we examined Akata cells, an EBV-positive B cell line derived from a Burkitt's lymphoma. As shown in Fig. 1C, phosphorylation of ATM and Chk2 was again observed upon induction of lytic replication. Furthermore, as shown in Fig. 1D, it should be noted that expression of the BZLF1 protein alone in Hela cells did not phosphorylate NBS1 Ser-343, Chk2 at Thr-68, and p53 at Ser-15 as judged by Western blotting, suggesting that expression of the BZLF1 protein itself cannot elicit DNA damage response checkpoint signaling pathways. Phosphorylated ATM Accumulates in Viral Replication Compartments After Induction of Lytic Replication—Many proteins involved in the DNA damage response accumulate in foci at sites of DSBs or abnormal DNA structures such as single-stranded DNA (33Rouse J. Jackson S.P. Science. 2002; 297: 547-551Crossref PubMed Scopus (578) Google Scholar). It has previously been demonstrated that ATM protein becomes associated with chromatin upon ionizing radiation, using a biochemical fractionation procedure in which a portion of the ATM pool was found to be resistant to detergent extraction after treatment with agents that cause DSBs (18Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (424) Google Scholar, 34Andegeko Y. Moyal L. Mittelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). EBV lytic DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins cluster and viral DNAs are synthesized (25Takagi S. Takada K. Sairenji T. Virology. 1991; 185: 309-315Crossref PubMed Scopus (37) Google Scholar). Lytic replication-induced Tet-BZLF1/B95-8 cells were extracted with 0.5%Triton X-100-mCSK buffer to solubilize DNA-unbound forms of viral or cellular proteins (35Fujita M. Ishimi Y. Nakamura H. Kiyono T. Tsurumi T. J. Biol. Chem. 2002; 277: 10354-10361Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). As shown in Fig. 2A, the BMRF1 and BALF2 viral replication proteins were colocalized in the nuclei after induction of lytic replication. The sites were completely coincided with the localized foci of newly synthesized viral DNA as judged by BrdUrd incorporation and FISH analyses (Fig. 2A, panels b and c). Thus, since the BALF2 or BMRF1 protein-localized sites represent loci of viral DNA synthesis, these were used as markers for viral replication compartments. We examined whether activated DNA damage responsive proteins accumulate in such foci after induction of lytic infection. As shown in Fig. 2B, in the lytic replication-induced cells, ATM phosphorylated at Ser-1981 was found to be resistant to detergent extraction and became colocalized with viral DNAs in the replication compartments, strongl
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