Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment *

Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 9, Issue of March 4, pp. 8156–8163, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment* Received for publication, October 6, 2004, and in revised form, December 15, 2004 Published, JBC Papers in Press, December 15, 2004, DOI 10.1074/jbc.M411405200 Ayumi Kudoh‡§ , Masatoshi Fujita‡ , Lumin Zhang‡§, Noriko Shirata‡, Tohru Daikoku‡, Yutaka Sugaya‡, Hiroki Isomura‡, Yukihiro Nishiyama§, and Tatsuya Tsurumi‡** From the ‡Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan, the Virology Division, National Cancer Center, Chuo-ku, Tokyo 104-0045, Japan, and the §Department of Virology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan related (ATR), respond to a variety of abnormal DNA structures When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or ar- and initiate signaling cascades leading to a DNA damage check- rest of cell-cycle progression, providing time for DNA point (1). ATM responds to the presence of DNA double-strand repair. Induction of the Epstein-Barr virus (EBV) lytic breaks (DSBs) induced by ionizing radiation (2). On the other program elicited a cellular DNA damage response, with hand, the ATR pathway can be stimulated by hydroxyurea, UV activation of the ataxia telangiectasia-mutated (ATM) light, and base-damaging agents that interfere with the move- signal transduction pathway. Activation of the ATM- ment of replication forks (3). The ATR pathway also responds Rad3-related (ATR) replication checkpoint pathway, in to DSBs, but more slowly than ATM (4). contrast, was minimal. The DNA damage sensor Mre11- A variety of checkpoint proteins have been identified as Rad50-Nbs1 (MRN) complex and phosphorylated ATM substrates for ATM and ATR kinases, including the checkpoint were recruited and retained in viral replication com- kinases Chk1 and Chk2, as well as H2AX (5) and p53 (2, 6). partments, recognizing newly synthesized viral DNAs as ATM phosphorylates Chk2 at several sites including Thr-68, abnormal DNA structures. Phosphorylated p53 also be- followed by Chk2 activation (6–9). Chk1 is phosphorylated at came concentrated in replication compartments and Ser-345 by ATR in response to UV and hydroxyurea, leading to physically interacted with viral BZLF1 protein. Despite a 3–5-fold increase in Chk1 activity (6, 7, 10). ATM is activated the activation of ATM checkpoint signaling, p53-down- by intermolecular autophosphorylation on Ser-1981 (11). Both stream signaling was blocked, with rather high S-phase Mre11 and Nbs1 are also targets of ATM and possibly ATR (10, CDK activity associated with progression of lytic infec- tion. Therefore, although host cells activate ATM check- 12–14). The MRN complex consisting of Mre11, Rad50, and point signaling with response to the lytic viral DNA Nbs1 has been proposed to facilitate ATM activation (15–17) synthesis, the virus can skillfully evade this host check- and recently demonstrated to function upstream of ATM acti- point security system and actively promote an S-phase- vation as a damage sensor, in addition to acting as an effector like environment advantageous for viral lytic replication. of ATM signaling (15, 18). ATM/ATR-initiated checkpoint signaling induces p53- dependent and p53-independent responses. The p53-dependent Eukaryotic cells exhibit a variety of physiological responses, cell cycle checkpoint features p21-mediated inactivation of including cell cycle arrest, activation of DNA repair and apo- Cdk2/cyclin E (19–21), while Chk2 inhibits Cdk2/cyclin E ac- ptosis, upon DNA damage. Sets of checkpoint proteins that tivity by phosphorylation of Cdk2 at Tyr-15, via down-regula- have been conserved with evolution are rapidly induced to tion of CDC25A phosphatase (4), in a p53-independent fashion. prevent replication or segregation of damaged DNA before re- Among Cdk2-targets, the Rb protein is most important for cell pair is completed. The related phosphatidylinositol 3-like ki- cycle progression and the checkpoint pathways result in its nases, ataxia telangiectasia-mutated (ATM) and ATM-Rad3- hypophosphorylation, leading to G or G /M cell cycle arrest. 1 2 The Epstein-Barr virus (EBV) is a human herpes virus that infects 90% of individuals. Primary EBV infection targets rest- * This work was supported by Grants-in-aid for Scientific Research ing B lymphocytes, inducing their continuous proliferation. In on Priority Areas from the Ministry of Education, Science, Sports, the B lymphoblastoid cell lines (LCL) only limited numbers of Culture and Technology of Japan (16017322 and 15390153, to T. T.). The costs of publication of this article were defrayed in part by the viral genes are usually expressed and there is no production of payment of page charges. This article must therefore be hereby marked virus particles, this being called latent infection. In the latent “advertisement” in accordance with 18 U.S.C. Section 1734 solely to state, EBV maintains its 170 kbp genome as complete, multiple indicate this fact. copies of plasmids that are synthesized only once in each S- ¶ Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. phase by the host cell replication machinery, following the ** To whom correspondence should be addressed: Division of Virol- rules of chromosome replication (22). ogy, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa- EBV-infected cell lines usually contain a small subpopula- ku, Nagoya 464-8681, Japan. Tel./Fax: 81-52-764-2979; E-mail: tion of cells that have switched spontaneously from a latent [email protected]. The abbreviations used are: ATM, ataxia telangiectasia-mutated; stage of infection into the lytic cycle. The mechanism of switch- EBV, Epstein-Barr virus; PBS, phosphate-buffered saline; h.p.i., h post- ing is not fully understood, but one of the first detectable induction; Gy, Gray; ATR, ATM-Rad3-related; DSB, double-stranded changes is expression of the BZLF1 immediate-early gene prod- break; FISH, fluorescence in situ hybridization; BrdUrd, bromode- uct. The BZLF1 protein, together with the other immediate- oxyuridine; MRN, Mre11-Rad50-Nbs1; mCSK butter, modified cy- toskelton butter; Pipes, 1,4-piperazinediethanesulfonic acid. early protein, BRLF1 protein, transactivates viral promoters 8156 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. DNA Damage Signaling during EBV Lytic Replication 8157 molecular mass proteins (180 kDa) or phosphorylated form of Rb pro- (23) and leads to an ordered cascade of viral early and late gene teins, gradient SDS-PAGE or SDS-7.5% PAGE (acrylamide, 72; bisacryl- expression. Early gene products include proteins involved in amide, 1), respectively, were applied. The proteins were then processed as viral DNA replication and DNA metabolism. The lytic phase of described previously (26). Detection of target proteins was with an en- EBV DNA replication is dependent on seven viral replication hanced chemiluminescence detection system (Amersham Biosciences). proteins: BZLF1, an oriLyt-binding protein; BALF5, a DNA Immunofluorescence—Cells were treated with 0.5% Triton X-100- polymerase; BMRF1, a polymerase processivity factor; BALF2, mCSK buffer (10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl ,1mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl a single-stranded DNA-binding protein; and BBLF4, BSLF1, 2 fluoride, 10 g/ml aprotinin, and 0.5% Triton X-100) for 10 min on ice, and BBLF2/3, predicted to be helicase-, primase-, and helicase followed by fixation with 70% methanol for 20 min on ice. The fixed cells primase-associated proteins, respectively (24). Viral lytic replica- were washed with PBS and blocked for 20 min in 10% normal goat tion occurs in discrete sites in nuclei, called replication compart- serum in PBS. Staining with a mouse monoclonal antibody to phospho- ments in which viral replication proteins are assembled (25). rylated Ser-1981 of ATM, mouse monoclonal antibodies to NBS1, We have previously demonstrated that induction of the EBV 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% lytic program results in inhibition of replication of cellular goat serum. Staining with a rabbit polyclonal antibody to BALF2, a DNA as well as explosive replication of viral DNA (26). The mouse monoclonal antibody to BMRF1 and a rabbit polyclonal antibody levels of p53 and CDK inhibitors remain unchanged through- to BZLF1 was carried out for1hat room temperature. Species-specific out the lytic infection, while the amounts of cyclin E/A and the secondary antibodies were applied for1hat room temperature. Alexa- hyperphosphorylated form of Rb increase as lytic infection 488 and Alexa-594, highly cross-absorbed secondary reagents, were progresses. The resultant S-phase-like cellular condition is purchased from Molecular Probes and slides were mounted in Vecta- shield (Vector Labs) and analyzed by fluorescence confocal microscopy. found to be essential for the transcription of viral immediate- Confocal fluorescence images were captured and processed using a early and early genes probably attributed to transcription fac- Radiance2000 Confocal System (Bio-Rad). All the primary antibodies tors such as E2F-1 and Sp1 expressed during S phase (27). were employed at 1:100 dilutions, and the secondary antibodies at 1:500 It is of interest to determine whether host cells can monitor dilutions. All washes after antibody incubations were performed with EBV lytic replication as DNA damage or abnormal DNA and, if 0.05% Tween-20 in PBS at room temperature. The specificity of the so, how EBV blocks the checkpoint signaling to avoid G or second antibodies and reliability of discrimination with fluorescent microscopy filters were tested. When cells were stained singly for either G /M cell cycle arrest and apoptosis. We have previously iso- antigen with inappropriate combinations of first and second antibodies, lated EBV latently infected Tet-BZLF1/B95-8 cells in which no fluorescence was observed and also no immunofluorescence was exogenous BZLF1 protein is conditionally expressed under the observed with alternate filters. control of a tetracycline-regulated promoter (26). Using this For staining the BrdUrd-incorporated DNA, cells were treated for 10 system, we show here for the first time that induction of EBV min with 2 N HCl containing 0.5% Triton X-100 to expose the incorpo- lytic replication elicits a cellular DNA damage response de- rated BrdUrd residues before blocking. The cells were washed and neutralized with 0.1 M sodium tetraborate (pH 9.0) for 5 min of incu- pendent on ATM. DNA damage sensor MRN complex and phos- bation. For BrdUrd staining, Alexa Fluor 488-conjugated anti-BrdUrd phorylated ATM are recruited to viral replication compart- mouse monoclonal antibody was used. ments, presumably recognizing newly synthesized viral DNAs Fluorescence in Situ Hybridization (FISH)—EBV BamHI-W frag- as abnormal DNA structures. However, the ATM checkpoint ment was labeled with Chroma Tide Alexa Fluor 594-5-dUTP (Molec- signaling was blocked at downstream of p53. Therefore, al- ular Probes, Inc.) and used for the detection of amplified EBV genomes. though EBV lytic replication elicits ATM-dependent DNA dam- At first, immunostaining was performed as described above and then refixed in 4% paraformaldehyde to cross-link bound antibodies. After age response, the virus can skillfully block the host response permeabilizing in 0.2% Triton X-100 (20 min on ice), cells were digested and actively promote an S-phase-like environment advanta- with RNase A, dehydrated in ethanol, air-dried, and immediately cov- geous for viral lytic replication. ered with the probe mixture containing 50% formamide in 2 SSC containing probe DNA (10 ng/l), 10% dextran sulfate, salmon sperm EXPERIMENTAL PROCEDURES DNA (0.1 g/l), and yeast tRNA (1 g/l). Probe and cells were simul- Cells—Tet-BZLF1/B95-8 cells, a marmoset B-cell line latently in- taneously heated at 94 °C for 4 min and incubated overnight at 37 °C. fected with EBV (26), and Tet-BZLF1/Akata cells, human EBV-positive After hybridization, specimens were washed at 37 °C with 50% form- Burkitt’s lymphoma cells (27), were maintained in RPMI medium sup- amide in 2 SSC (two times for 15 min each) and 2 SSC. Finally, cells plemented with 1 g/ml of puromycin, 250 g/ml of hygromycin B, and were equilibrated in PBS and mounted in vectashield (Vector Labora- 10% tetracycline-free fetal calf serum. To induce lytic EBV replication, tories, Inc.). a tetracycline derivative, doxycycline, was added to the culture medium Immunoprecipitation—Cells were lysed in 1 ml of EBC lysis buffer at a final concentration of 2 g/ml. B95-8 cells were cultured in RPMI (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40) containing medium supplemented with 10% fetal calf serum. 100 mM sodium fluoride, 2 mM sodium vanadate, and protease inhibitor Antibodies—Primary antibodies were purchased from Cell Signaling mixture (Sigma; 25 l/ml), and then sonicated. The lysates were cen- (ATM-S1981, Chk1, Chk1-S345, Chk2-T68, p95/NBS1-S343, and p53- trifuged at 18,000  g for 20 min at 4 °C, and immunoprecipitation was S15), Genetex (ATM-2C1), BD Transduction Laboratories (Cdk1, Cdk2, performed using 1 mg of the supernatant and 5 g of anti-BZLF1 Chk2, Mre11, and Nbs1), Oncogene (p53, p21 and MDM2), Santa Cruz protein-specific IgG-beads or control rabbit IgG beads, with gentle Biotechnology (cyclin A-C19, cyclin B1-GCN1, cyclin E -M20, and E2F1- rocking for1hat4 °C. Ternary protein A-antibody-antigen complexes KH95), Upstate (H2AX-S139), Chemicon (EBV BMRF1-R3) and Argene were collected by centrifugation and washed three times with NET-gel (EBV BRLF1–8C12). Rabbit polyclonal antibodies to BZLF1, BALF2, buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Nonidet P-40, and BBLF2/3, and BALF5 proteins were prepared as described (26). Highly 1mM EDTA). The immunoprecipitates were subjected to SDS- cross-absorbed secondary reagents for dual-color detection (Alexa-488 10%PAGE followed by immunoblotting analyses. and 594) were from Molecular Probes. In Vitro Protein Kinase Assay—Cells were washed with ice-cold PBS Protein Preparation—Tet-BZLF1/B95-8, Tet-BZLF1/Akata, and and then lysed with EBC lysis buffer containing 100 mM sodium fluo- B95-8 cells were harvested at the indicated times post-treatment with ride, 2 mM sodium vanadate, and multiple protease inhibitors (Sigma; doxycycline, washed with phosphate-buffered saline (PBS), and treated 25 l/ml) for 5 min on ice followed by centrifugation at 18,000  g for 20 with lysis buffer (0.02% SDS, 0.5% Triton X-100, 300 mM NaCl, 20 mM min at 4 °C. For kinase assays 200 g of aliquots of protein extracts Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol) for 20 min on ice. were incubated with 1 g of each antibody in a final volume of 500 l for Multiple protease inhibitors (Sigma; 25l/ml), 200 M sodium vana- 1 h at 4 °C to precipitate cyclin-CDK complexes: C-19 against cyclin A, date, and 20 mM sodium fluoride were added to the buffer. Samples M-20 against cyclin E and GNS1 against cyclin B. Immune complexes were centrifuged at 18,000  g for 10 min at 4 °C, and clarified cell absorbed onto protein A-Sepharose were washed twice with ice-cold extracts were assayed for protein concentration using a Bio-Rad kit. NET-gel buffer, and then once with basic kinase buffer (200 mM Tris- Immunoblot Analysis—Equal amounts of proteins (2050 g) were HCl, pH 7.5, 10 mM MgCl ,1mM EDTA, 1 mM dithiothreitol). The loaded into each lane for SDS-10% polyacrylamide (acrylamide, 29.2; immunoprecipitates were resuspended in 24 l of kinase buffer (200 mM bisacrylamide, 0.8) gel electrophoresis (SDS-PAGE). To separate high Tris-HCl, pH 7.5, 10 mM MgCl ,1mM EDTA, 1 mM dithiothreitol, 50 M 2 8158 DNA Damage Signaling during EBV Lytic Replication ATP containing 5 Ci of [- P]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 lof5 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 g/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  10 cells and quantified. Dot-blot hybridization was performed, and quantification of the copy numbers of viral genome per cell were determined as described previously (26). RESULTS 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 pro- teins after induction of lytic replication with doxycycline were described previously (26). BZLF1 protein became detectable 4 h post-induction (h.p.i.) (26) and reached a plateau at 48 h.p.i. (Fig. 1A). The other immediate-early protein, the BRLF1 pro- tein also appeared at 6 h.p.i (26) 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-associ- ated 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 path- way 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 (11). 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 phos- phorylate Thr-68 on Chk2, which is required for its activation (9, 28). Immunoblotting with anti-Chk2 Thr-68-specific anti- body 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 (29, 30), was also exam- ined. 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 FIG.1. Activation of DNA damage responsive proteins upon expression levels of p53 protein throughout lytic infection, in induction of lytic replication. Tet-BZLF1/B95-8 cells or B95-8 cells agreement with our previous observation (26). were cultured in the presence of 2 g/ml of doxycycline, harvested at the indicated times. Equal amounts of proteins for each sample (20  50 g) It should be noted that not only activated ATM but also ATR were applied for immunoblot analyses. A, expression profile of EBV kinases phosphorylate Chk2 kinase at Thr-68 and up-regulate lytic proteins in lytic program-induced Tet-BZLF1/B95-8 cells. B, ex- its activity (9, 28). Phosphorylation of histone H2AX or p53 at pression levels of ATM autophosphorylated at Ser-1981, ATM, Chk2 Ser-15 is also carried out by ATM/ATR kinases (31, 32). There- phosphorylated at Thr-68, Chk2, Chk1 phosphorylated at Ser-345, Chk1, H2AX phosphorylated at Ser-139 (H2AX), p53 phosphorylated fore, EBV lytic replication could activate the ATM, ATR, or at Ser-15, and p53, were determined by immunoblotting. C, the Tet- both kinases. The ATR kinase responds primarily to DNA BZLF1/Akata cells treated with 2 g/ml of doxycycline were harvested replication stress during S phase (3). It can also respond to at indicated times. Clarified cell lysates were prepared and applied for DSBs if within the S phase, but less efficiently than ATM. In Western blot analyses with each antibody as indicated. D, the Tet- BZLF1/Hela cells treated with 2 g/ml of doxycycline were harvested at contrast to Chk2, phosphorylation of Chk1 at Ser-345 is known indicated times. Clarified cell lysates were prepared and applied for to be carried out mainly by ATR kinase, leading to its activa- Western blot analyses with each antibody as indicated. HU, cells were tion (10). Therefore, we examined Chk1 phosphorylation at treated with 5 mM of hydroxyurea for 12 h and harvested. IR, cells were Ser-345 in lytic replication-induced B95-8 cells, but no signifi- exposed to -radiation with 10 Gy and harvested 4 h post-treatment. cant phosphorylation was observed (Fig. 1B). Treatment of cells with hydroxyurea, a well-studied activator of the replica- Thus, we conclude that EBV lytic replication elicits activation tion checkpoint, clearly induced Chk1 phosphorylation (Fig. of ATM DNA damage checkpoint signaling rather than the 1B), showing the ATR/Chk1 pathway to be intact in the cells. ATR pathway that responds to replication stress. DNA Damage Signaling during EBV Lytic Replication 8159 FIG.2. Subcellular localization of ATM Ser-1981 after induc- tion of lytic replication. A, architecture of viral replication compart- ments. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 g/ml doxycycline for 14 h and newly synthesized DNAs were labeled with 10 M BrdUrd for 1 h prior to harvest. Cells were treated with 0.5% Triton FIG.3. NBS1 and Mre11 proteins constituting the MRN com- X-100-mCSK buffer and were then fixed with methanol viral replication plex are recruited to EBV replication compartments. A, Tet- proteins (BMRF1 and BALF2 proteins), newly synthesized DNA, or BZLF1/B95-8 cells were cultured in the presence of 2 g/ml of doxycy- viral DNA was visualized by immunofluorescence, BrdUrd staining, or cline for the indicated times, and expression levels of Nbs1 and Mre11 FISH analyses, respectively. Right panels are merged images. B, sub- were analyzed by immunoblotting with each antibody as indicated. B, nuclear localization of phosphorylated ATM. Tet-BZLF1/B95-8 cells recruitment of Nbs1 and Mre11 constituting the MRN complex to viral were cultured in the absence (panel a) or presence of 2 g/ml of doxy- replication compartments. Tet-BZLF1/B95-8 cells were cultured in the cycline and harvested at 14 h.p.i. (panels b and d) or 24 h.p.i. (panels c absence (panels a, b, and d) or presence (panels c and e)of2 gof and e). Cells were treated with 0.5% Triton X-100-mCSK buffer. The doxycycline/ml and harvested at 14 h.p.i. Cells were treated with 0.5% nonionic-detergent-extracted cells were then fixed with methanol. Pro- Triton X-100-mCSK buffer, fixed with methanol and coimmunostained teins (BALF2 protein and ATM phosphorylated at Ser-1981) and viral with antibodies to BALF2 protein and Nbs1 protein (panels a, b, and c) DNA was visualized by immunofluorescence and FISH analyses, re- or Mre11 protein (panels d and e). Panel b, Tet-BZLF1/B95-8 cells were spectively. Right panels are merged images. exposed to 10 Gy  irradiation and harvested 4 h post-treatment. Cells were processed in parallel with the lytic program-induced cells. Right panels are merged images. To ascertain whether lytic replication elicits ATM/Chk2 DNA damage checkpoint signaling in other EBV-latently in- fected B cells, we examined Akata cells, an EBV-positive B cell thesis, these were used as markers for viral replication com- line derived from a Burkitt’s lymphoma. As shown in Fig. 1C, partments. We examined whether activated DNA damage re- phosphorylation of ATM and Chk2 was again observed upon sponsive proteins accumulate in such foci after induction of induction of lytic replication. Furthermore, as shown in Fig. lytic infection. As shown in Fig. 2B, in the lytic replication- 1D, it should be noted that expression of the BZLF1 protein induced cells, ATM phosphorylated at Ser-1981 was found to be alone in Hela cells did not phosphorylate NBS1 Ser-343, Chk2 resistant to detergent extraction and became colocalized with at Thr-68, and p53 at Ser-15 as judged by Western blotting, viral DNAs in the replication compartments, strongly suggest- suggesting that expression of the BZLF1 protein itself cannot ing that activated ATM recognizes and binds to newly synthe- elicit DNA damage response checkpoint signaling pathways. sized viral DNA. In contrast, phosphorylated ATM was not Phosphorylated ATM Accumulates in Viral Replication Com- observed in the latently infected cells (Fig. 2B, panel a). partments After Induction of Lytic Replication—Many proteins The Nbs1 and Mre11 Proteins Constituting the MRN Com- involved in the DNA damage response accumulate in foci at plex Accumulate in Viral Replication Foci—The MRN complex consisting of Mre11, Rad50, and Nbs1 has been suggested to sites of DSBs or abnormal DNA structures such as single- stranded DNA (33). It has previously been demonstrated that act as a damage sensor (14, 18), facilitating ATM activation (15). After ionizing radiation, relocalization of the MRN com- ATM protein becomes associated with chromatin upon ionizing radiation, using a biochemical fractionation procedure in which plex at sites of damage is readily observed in detergent-ex- tracted cells (36) and independent of ATM and a portion of the ATM pool was found to be resistant to detergent H2AX (18, 31, extraction after treatment with agents that cause DSBs (18, 36). As shown in Fig. 3A, the levels of Mre11 and Nbs1 were 34). EBV lytic DNA replication occurs at discrete sites in nu- constant throughout the lytic infection, unlike the case for clei, called replication compartments, where viral replication adenovirus infection which degrades the MRN complex (18, proteins cluster and viral DNAs are synthesized (25). Lytic 37). Activated ATM also phosphorylates Ser-343 on Nbs1 (12, replication-induced Tet-BZLF1/B95-8 cells were extracted with 13). As shown in Fig. 3A, increase in levels of phosphorylated 0.5%Triton X-100-mCSK buffer to solubilize DNA-unbound form of Nbs1 at Ser-343 was observed, this becoming detectable forms of viral or cellular proteins (35). As shown in Fig. 2A, the after12 h.p.i. Next, we assessed the effect of induction of EBV BMRF1 and BALF2 viral replication proteins were colocalized lytic replication on the localization of Mre11 and Nbs1 by in the nuclei after induction of lytic replication. The sites were immunofluorescence after detergent extraction (Fig. 3B). No completely coincided with the localized foci of newly synthe- staining with Mre11-specific or Nbs1-specific antibodies was sized viral DNA as judged by BrdUrd incorporation and FISH observed in the detergent-treated latently infected cells (Fig. analyses (Fig. 2A, panels b and c). Thus, since the BALF2 or 3B, panels a and d). As shown in Fig. 3B, panel b, ionizing BMRF1 protein-localized sites represent loci of viral DNA syn- radiation resulted in distinct staining of Nbs1 in the nuclei, 8160 DNA Damage Signaling during EBV Lytic Replication FIG.4. S-phase-like environment after induction of lytic replication. Tet-BZLF1/B95-8 or B95-8 cells were cultured in the presence of 2 g/ml of doxycycline and harvested at the indicated times. Clarified cell lysates were prepared, and the proteins were analyzed by Western blotting cip1/waf1 with specific antibodies. A, expression levels of p21 and MDM2 after treatment with doxycycline in Tet-BZLF1/B95-8 cells and B95-8 cells. IR, Tet-BZLF1/B95-8 cells were exposed to 10 Gy  irradiation and harvested 4 h post-treatment. B, acceleration of proteasomal degradation of p53 in lytic phase of EBV replication. Tet-BZLF1/B95-8 cells were cultured in the presence (Dox ) or absence (Dox )of2 g/ml of doxycycline. Proteasome inhibitor, 20 M MG132 plus 0.1% Me SO (MG132), or 0.1% Me SO as control (MG132) was added to the culture at 16 h.p.i., and 2 2 cells were harvested at 24 h.p.i. C, accumulation of E2F-1 accompanied by hyperphosphorylation of Rb after induction of the lytic program in Tet-BZLF1/B95-8 cells. The proteins were separated by SDS-7.5% PAGE (for RB) or SDS-10% PAGE (for others) and analyzed by Western blotting with antibodies as indicated. The slower migrating bands are hyperphosphorylated forms of the Rb protein (ppRB and pRB). The faster migrating band is the hypophosphorylated form of the Rb protein (RB). D, elevation of cyclin A- or cyclin E-associated CDK activity during EBV lytic replication. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 g/ml of doxycycline and harvested at the indicated times. Equal amounts of whole cell extracts were prepared and used for immunoprecipitation (IP) with antibodies against each of the indicated cyclins, and then kinase assays were performed using histone H1 as substrate, as described under “Experimental Procedures.” Lower panel shows the densitometric analysis of cyclin A-, cyclin E-, and cyclin B-associated CDK activities. The signal intensity was quantified with an Image Guider (BAS2500, Fuji film). indicating that the MRN complex is activated and retained in throughout lytic replication (Fig. 1B). Therefore, we exam- the damaged sites. Upon induction of lytic replication, Mre11 ined expression levels of p53-transcriptional targets such as cip1/waf1 and Nbs1 proteins became resistant to detergent treatment p21 , a cyclin-dependent kinase inhibitory protein, and and colocalized predominantly in viral replication compart- MDM2, a ubiquitin protein ligase, for p53 (Fig. 4A). When cip1/waf1 ments represented by the BALF2 staining (Fig. 3B, panels c Tet-BZLF1/B95-8 cells were irradiated, the levels of p21 and e). Once the pools of endogenous Mre11 and Nbs1 protein and MDM2 were elevated remarkably in response. In contrast, were concentrated in this way, the associated fluorescence be- the level of MDM2 ubiquitin ligase came down with progres- cip1/waf1 came resistant to extraction with a mild detergent-containing sion of lytic replication, and the level of p21 was low and extraction buffer, indicating that the Mre11 and Nbs1 proteins almost unchanged throughout the lytic replication. These sug- became not only redistributed to, but also retained within the gest that EBV lytic program possesses a defense system to close vicinity of newly synthesized viral DNA. It is likely that prevent p53 downstream signaling. The levels of p53-independ- kip1 ink4a the MRN complex recognizes newly synthesized viral genomic ent CDK inhibitors such as p27 and p16 were also DNA in the replication compartments as abnormal DNA struc- unchanged (data not shown). These observations suggest that tures and binds to them. p53 downstream signaling is blocked during EBV lytic infection Inhibition of p53 Downstream Target Gene Expression after despite the appearance of phosphorylated p53. Induction of EBV Lytic Infection—As described above, induc- Next, the relative abundance of p53 was analyzed by West- tion of EBV lytic replication elicited phosphorylation of ATM, ern blot in the presence of the chemical proteasome inhibitor, Nbs1, H2AX, Chk2, and p53. Phosphorylation of p53 at Ser-15 MG132. As shown in Fig. 4B, the amounts of p53 in EBV in response to DNA damage usually correlates with both accu- latently infected cells were almost constant in the absence and mulation of total p53 protein as well as with the ability of p53 presence of MG132. By contrast, MG132 stabilized p53, result- to transactivate downstream target genes in wild-type cells ing in comparative accumulation of p53 in the lytic infection- (38). However, the level of p53 here proved to be constant induced cells. Thus, it was clearly demonstrated that p53 turn- DNA Damage Signaling during EBV Lytic Replication 8161 over is regulated by proteasome degradation after induction of the lytic infection. These results are intriguing because the degradation of p53 via proteasome appears to be one of the mechanisms that explain why the phosphorylated form of p53 at Ser-15 cannot transactivate its downstream factors such as p21 and MDM2. Since the level of MDM2 ubiquitin ligase came down with progression of lytic replication, degradation of p53 might be mediated by a direct interaction and recruitment of ubiquitin ligase activity other than MDM2. Activation of S-phase-promoting CDK Activity throughout Lytic Infection—As shown in Fig. 4C, we observed that the levels of cyclin E and cyclin A. continued to be elevated, whereas the level of cyclin B was constant during the lytic replication, con- firming our previous report (26). These data strongly suggest that S-phase-promoting CDK, namely cyclin A/E-Cdk2, is acti- vated during lytic infection. In fact, as shown in Fig. 4D, cyclin E- and cyclin A-associated CDK activity increased as lytic replica- tion progressed, whereas cyclin B-associated kinase activity was unchanged and rather down-regulated at 48 h.p.i. Slow migrat- ing hyperphosphorylated Rb proteins accumulated with progres- sion of lytic infection (Fig. 4C), probably because of elevated cyclin E- and A-associated CDK activity. Also, the levels of E2F-1 increased with progression of lytic infection (Fig. 4C). Hyperphos- phorylation of Rb may result in release of an active form of E2F-1, which binds to its own binding site on E2F-1 promoters and enhances expression (39). Thus, these observations clearly indi- cate that EBV lytic replication occurs in an S phase-like cellular environment regardless of elicitation of the ATM DNA damage response. p53 Physically Interacts with EBV BZLF1 Protein in Vivo and Is Recruited to Viral Replication Compartments—Zhang et al. (40) has previously reported that the BZLF1 protein inhibits transactivation activity of p53, likely through the direct inter- action demonstrated using an adenovirus overexpression sys- FIG.5. Phosphorylated p53 interacts with the BZLF1 protein. tem. To confirm actual protein-protein interaction in vivo and A, immunoprecipitation of the BZLF1 protein with an anti-p53 specific resolve the paradox between induction of ATM DNA damage antibody. Tet-BZLF1/B95-8 cells were untreated or treated with 2 g/ml doxycycline and harvested at 48 h.p.i. Clarified lysates were response and inhibition of p53 downstream events, immuno- prepared and subjected to immunoprecipitation (IP) analysis with anti- precipitation analyses with an anti-BZLF1 protein-specific an- BZLF1 IgG or control rabbit IgG beads. Aliquots of the immunoprecipi- tibody were performed with lytic replication-induced B95-8 cell tated proteins and lysates (Input) were analyzed by Western blotting extracts. As shown in Fig. 5A, the antibody immunoprecipi- with anti-p53 monoclonal IgG, anti-p53Ser-15 polyclonal IgG, or anti- BZLF1 polyclonal IgG. B, colocalization of p53 with the BZLF1 protein tated phosphorylated p53. Thus, the physical interaction be- in viral replication compartments. Tet-BZLF1/B95-8 cells were cultured tween the BZLF1protein and the phosphorylated form of p53 in the absence (panels a and c) or presence (panels b and d)of2 gof was confirmed not only in the overexpression system (40) but doxycycline/ml and harvested at 24 h.p.i. Cells were treated with 0.5% also in the lytic phase-induced B95-8 cells. Triton X-100-mCSK buffer, fixed with methanol, and coimmunostained with the indicated antibodies. Panels a and b show images of BZLF1 Next, we examined the localization of p53 using a specific protein and p53. Panels c and d show images of BMRF1 protein and p53 antibody to p53 or phospho-Ser-15 p53. In latently infected phosphorylated at Ser-15. Right panels are merged images. cells p53 was sensitive to detergent treatment and no staining with a p53-specific antibody was observed (Fig. 5B, panels a and c). In contrast, the p53-associated fluorescence became phosphorylation of target proteins of ATM kinase activity such resistant to extraction by a mild detergent-containing buffer as Chk2 at Thr-68, ATM at Ser-1981, and Nbs1 at Ser-343, the after induction of lytic replication. Thus, p53 became re- expression levels of viral replication proteins were not affected tained in the nuclei and colocalized with the BZLF1 protein at all (Fig. 6A). These data suggest that a caffeine-sensitive in the replication compartments. Also, p53 phosphorylated at kinase is involved in the checkpoint signaling evoked during Ser-15 was recruited to viral replication compartments. the EBV lytic infection, while ATM signaling is not absolutely Taken together, the results clearly demonstrated that phos- required for lytic replication. Moreover, in order to clarify the phorylated p53 was redistributed to viral replication com- effects of caffeine on EBV genome synthesis, Tet-BZLF1/B95-8 partments, associated with BZLF1 protein via physical cells were treated with doxycycline in the presence or absence interaction. of the compound. Total DNA was extracted from the cells, and Inhibition of ATM-dependent DNA Damage Response In- EBV DNA-specific signals were quantitated. As shown in Fig. duced by EBV Lytic Replication Does Not Affect Viral Lytic 6B, the copy number of the viral DNA was amplified up to more Replication—Phosphorylation states of ATM DNA damage re- than 1500 copies per cell after 48 h.p.i. in the absence of sponsive proteins and expression levels of viral lytic proteins caffeine. Inhibition by caffeine of ATM-dependent checkpoint during lytic infection with EBV were examined in the presence activation induced by EBV lytic replication did not affect viral or absence of caffeine, a dose that inhibits the kinase activity of lytic replication at all. It is likely that since ATM DNA damage both ATM and ATR in vitro (41). Although caffeine treatment signaling induced by the lytic replication is blocked at least at of lytic program-induced Tet-BZLF1/B95-8 cells abrogated the level of p53 by the BZLF1 protein, inhibition of ATM/ATR 8162 DNA Damage Signaling during EBV Lytic Replication FIG.6. Inhibition by caffeine of ATM DNA damage checkpoint activa- tion induced by the lytic replication. A, lytic replication was induced in Tet- BZLF1/B95-8 cells with doxycycline in the presence or absence of 5 mM caffeine and harvested at indicated times. In parallel, uninduced Tet-BZLF1/B95-8 cells were also cultured in the presence of caffeine as a control. Clarified cell lysates were pre- pared, and the proteins were separated by SDS-10% PAGE and analyzed by Western blotting with specific antibodies. B, Tet- BZLF1/B95-8 cells were cultured with doxycycline (2 g/ml) in the presence (E) or absence (●)of5mM caffeine. Cells were harvested at the indicated times, and vi- ral DNA synthesis was determined by slot blot assay as described under “Experi- mental Procedures.” kinase activity by caffeine might have almost no effect on viral phorylation of Ser-345 on Chk1 (Fig. 1B). Further, recruitment lytic replication. We conclude that ATM signaling is not abso- of ATR to EBV replication compartments as judged by confocal lutely required for lytic replication. immunostaining analyses was not observed (data not shown). The ATR kinase responds primarily to chromosomal DNA rep- DISCUSSION lication stress during S phase (3). Taken together, our results It has been clearly demonstrated here that EBV lytic repli- indicate that EBV lytic replication preferentially activates the cation indeed induced the ATM DNA damage response, which ATM DNA damage response. At 24 h after lytic induction, some was blocked through the interaction between the BZLF1 pro- cells exhibited BrdUrd staining throughout nuclei. We could tein and p53. Also, activated Chk2 is known to inhibit Cdk2/ not observe any viral replication compartments stained with cyclin E or A activity by phosphorylation of Cdk2 at Tyr-15, via specific antibodies to the BMRF1 or the BALF2 lytic proteins in down-regulation of CDC25A phosphatase (4), in a p53-inde- such cells, suggesting that EBV lytic replication might not pendent fashion. Although Chk2 was phosphorylated by ATM occur in S-phase cells in which chromosomal DNA replication as shown in Fig. 1, cyclin A- and E-associated CDK2 activity has already started. If EBV lytic replication had arrested fork was increased with the progression of lytic replication. It has movements of chromosomal DNA replication, ATR DNA dam- been very recently reported that EBNA3a, one of viral proteins age checkpoint could be activated. Further experiments are expressed during latent infection, interacts with Chk2 to dis- needed to clarify this point. rupt G2/M checkpoint (42), suggesting inhibition of p53-inde- The expression level of E2F-1 is elevated as lytic replication pendent DNA damage signaling pathway. Since the G -or progresses. Although the precise mechanism remains to be G /M-arrested state or apoptosis following DNA damage check- determined, maintaining low levels of CDK inhibitors during point signaling would be unsuitable for viral lytic replication, lytic infection might result in accumulation of the hyperphos- EBV would create the cell to become an S-phase-like condition. phorylated form of Rb protein by cyclin E and A associated ATM and the MRN complex function in a common pathway kinase activity. Phosphorylation of the Rb protein leads to (13), and the MRN complex can function to activate ATM as a release from its inhibitory effects on E2F-1 as transcription damage sensor, in addition to acting as an effector of ATM factor (43), thereby deregulating E2F-1 in favor of an S-phase signaling (18). Clustering of ATM, Mre11, and Nbs1 proteins to environment. E2F-1 can transactivate not only the cyclin E and the EBV replication compartments from early stages of lytic cyclin A genes but also its own expression. Alternatively, it has infection strongly suggests that these damage sensors recog- been very recently reported that the BZLF1 protein by itself nize newly synthesized viral DNAs as abnormal DNA struc- activates E2F-1, cyclin E, and Cdc25A involved in cell cycle tures. Although ATM/ATR kinase phosphorylates histone progression in telomerase-immortalized human keratinocytes H2AX and p53 at Ser-15, ATR kinase activity predominantly and primary tonsil keratinocytes (44) and further that the phosphorylates Chk1 at Ser-345 leading to increased Chk1 activity (6, 7, 10). Surprisingly, we could not detect any phos- other immediate-early transactivator, BRLF1 protein, can in- DNA Damage Signaling during EBV Lytic Replication 8163 3. Osborn, A. J., Elledge, S. J., and Zou, L. (2002) Trends Cell Biol. 12, 509–516 duce contact-inhibited, quiescent human fibroblasts to enter 4. Zhou, B. B., and Elledge, S. J. (2000) Nature 408, 433–439 the S phase with dramatic increase in the level of E2F-1 (45). 5. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., Also, activated ATM or Chk2 phosphorylate and activate E2F-1 and Bonner, W. M. (2000) Curr. Biol. 10, 886–895 6. Kastan, M. B., and Lim, D. S. (2000) Nat. Rev. Mol. Cell. Biol. 1, 179–186 in response to DNA damage (46, 47) and thus the fact that 7. Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas, J. (2001) Nature inhibition of ATM kinase activity by caffeine treatment re- 410, 842–847 8. Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K., and Elledge, S. J. duced the level of E2F-1 (data not shown) is in line with the (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10389–10394 literature. 9. Melchionna, R., Chen, X. B., Blasina, A., and McGowan, C. H. (2000) Nat. Cell Infection with an E4-deleted adenovirus results in synthesis Biol. 2, 762–765 10. Zhao, H., and Piwnica-Worms, H. (2001) Mol. Cell. Biol. 21, 4129–4139 of end-joined large viral genomes that are recognized as abnor- 11. Bakkenist, C. J., and Kastan, M. B. (2003) Nature 421, 499–506 mal DNA structures by the MRN complex, leading to activation 12. Gatei, M., Young, D., Cerosaletti, K. M., Desai-Mehta, A., Spring, K., Kozlov, of cell cycle checkpoints. As a result, ATM damage response S., Lavin, M. F., Gatti, R. A., Concannon, P., and Khanna, K. (2000) Nat. Genet. 25, 115–119 signaling is activated and Chk1, Chk2, 53BP1, p53, Nbs1 and 13. Wu, X., Ranganathan, V., Weisman, D. S., Heine, W. F., Ciccone, D. N., ATM are phosphorylated (18, 37). Similarly, in the case of EBV, O’Neill, T. B., Crick, K. E., Pierce, K. A., Lane, W. S., Rathbun, G., Living- intermediate replication products in lytic phase are thought to ston, D. M., and Weaver, D. T. (2000) Nature 405, 477–482 14. D’Amours, D., and Jackson, S. P. (2002) Nat. Rev. Mol. Cell. Biol. 3, 317–327 be large head-to-tail concatemers with branched structures 15. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L., and Shiloh, Y. that might be generated by homologous recombination coupled (2003) EMBO J. 22, 5612–5621 with replication events (48, 49). In homologous recombination 16. Usui, T., Ogawa, H., and Petrini, J. H. (2001) Mol. Cell 7, 1255–1266 17. Williams, B. R., Mirzoeva, O. K., Morgan, W. F., Lin, J., Dunnick, W., and repair the MRN complex, most likely with help of other nucle- Petrini, J. H. (2002) Curr. Biol. 12, 648–653 ase, might resects the DNA to provide ssDNA overhangs nec- 18. Carson, C. T., Schwartz, R. A., Stracker, T. H., Lilley, C. E., Lee, D. V., and essary for DNA pairing and strand exchange, since the MRN Weitzman, M. D. (2003) EMBO J. 22, 6610–6620 19. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, complex is known to possess exonuclease activity. The Rad50 J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell subunit of MRN has ATPase activity that is believed to facili- 75, 817–825 tate DNA unwinding. Thus, such higher order intermediate 20. Gu, Y., Turck, C. W., and Morgan, D. O. (1993) Nature 366, 707–710 21. Dulic, V., Kaufmann, W. K., Wilson, S. J., Tlsty, T. D., Lees, E., Harper, J. W., viral genome structures might be recognized and made by the Elledge, S. J., and Reed, S. I. (1994) Cell 76, 1013–1023 MRN complex. We speculate that this is the case and that the 22. Yates, J. L., and Guan, N. (1991) J. Virol. 65, 483–488 MRN complex then modifies the DNA to create a platform for 23. Flemington, E. K., Goldfeld, A. E., and Speck, S. H. (1991) J. Virol. 65, 7073–7077 ATM and other signaling factors (14). When viral lytic DNA 24. Fixman, E. D., Hayward, G. S., and Hayward, S. D. (1995) J. Virol. 69, replication is blocked by the addition of phosphonoacetic acid 2998–3006 (PAA), a herpes virus DNA polymerase specific inhibitor, viral 25. Takagi, S., Takada, K., and Sairenji, T. (1991) Virology 185, 309–315 26. Kudoh, A., Fujita, M., Kiyono, T., Kuzushima, K., Sugaya, Y., Izuta, S., prereplicative sites represented by staining of the BALF2 pro- Nishiyama, Y., and Tsurumi, T. (2003) J. Virol. 77, 851–861 tein are formed (50). Although viral lytic immediate-early and 27. Kudoh, A., Daikoku, T., Sugaya, Y., Isomura, H., Fujita, M., Kiyono, T., Nishiyama, Y., and Tsurumi, T. (2004) J. Virol. 78, 104–115 early proteins were expressed even in the presence of PAA, no 28. Ahn, J. Y., Schwarz, J. K., Piwnica-Worms, H., and Canman, C. E. (2000) accumulation of synthesized viral DNA, Nbs1, and phosphoryl- Cancer Res. 60, 5934–5936 ated ATM to the prereplicative sites was observed (data not 29. Yazdi, P. T., Wang, Y., Zhao, S., Patel, N., Lee, E. Y., and Qin, J. (2002) Genes shown). This result also supports the idea that newly synthe- Dev. 16, 571–582 30. Falck, J., Petrini, J. H., Williams, B. R., Lukas, J., and Bartek, J. (2002) Nat. sized viral DNA is recognized by cellular DNA damage sensors. Genet. 30, 290–294 During wild-type adenovirus infection, the E1b55k-E4orf6 31. Celeste, A., Fernandez-Capetillo, O., Kruhlak, M. J., Pilch, D. R., Staudt, D. W., Lee, A., Bonner, R. F., Bonner, W. M., and Nussenzweig, A. (2003) complex degrades the MRN complex, preventing signaling Nat. Cell Biol. 5, 675–679 through both ATM and ATR pathways (18, 37). Also, the com- 32. Tibbetts, R. S., Brumbaugh, K. M., Williams, J. M., Sarkaria, J. N., Cliby, plex could also function in the regulation of p53 levels by W. A., Shieh, S. Y., Taya, Y., Prives, C., and Abraham, R. T. (1999) Genes Dev. 13, 152–157 degradation of p53 through ubiquitination (51). Thus, adenovi- 33. Rouse, J., and Jackson, S. P. (2002) Science 297, 547–551 ruses appear to have evolved double-check mechanisms to 34. Andegeko, Y., Moyal, L., Mittelman, L., Tsarfaty, I., Shiloh, Y., and Rotman, G. block cell cycle checkpoint signaling pathways. Unlike the case (2001) J. Biol. Chem. 276, 38224–38230 35. Fujita, M., Ishimi, Y., Nakamura, H., Kiyono, T., and Tsurumi, T. (2002) with adenoviruses, lytic replication of EBV does not degrade J. Biol. Chem. 277, 10354–10361 the MRN complex since the levels of Mre11 and Nbs1 were 36. Mirzoeva, O. K., and Petrini, J. H. (2001) Mol. Cell. Biol. 21, 281–288 constant throughout the lytic replication (Fig. 3A). However, 37. Stracker, T. H., Carson, C. T., and Weitzman, M. D. (2002) Nature 418, 348–352 the BZLF1 protein binds to phosphorylated p53, preventing its 38. Siliciano, J. D., Canman, C. E., Taya, Y., Sakaguchi, K., Appella, E., and transcriptional activity (40). p53 turnover is also regulated by Kastan, M. B. (1997) Genes Dev. 11, 3471–3481 proteasome degradation even after induction of lytic infection 39. Johnson, D. G., Ohtani, K., and Nevins, J. R. (1994) Genes Dev. 8, 1514–1525 40. Zhang, Q., Gutsch, D., and Kenney, S. (1994) Mol. Cell. Biol. 14, 1929–1938 (Fig. 4B). As shown in Fig. 4A, the level of MDM2 ubiquitin 41. Sarkaria, J. N., Busby, E. C., Tibbetts, R. S., Roos, P., Taya, Y., Karnitz, L. M., ligase was reduced with progression of lytic replication. There- and Abraham, R. T. (1999) Cancer Res. 59, 4375–4382 fore, degradation of p53 might be mediated by direct interac- 42. Krauer, K. G., Burgess, A., Buck, M., Flanagan, J., Sculley, T. B., and Gabri- elli, B. (2004) Oncogene 23, 1342–1353 tion and recruitment of protein(s) with ubiquitin ligase activ- 43. Nevins, J. R. (1992) Science 258, 424–429 ity, besides MDM2. It is likely that unidentified viral protein(s) 44. Mauser, A., Holley-Guthrie, E., Zanation, A., Yarborough, W., Kaufmann, W., might affect cellular regulators of p53 stability like the adeno- Klingelhutz, A., Seaman, W. T., and Kenney, S. (2002) J. Virol. 76, 12543–12552 virus E1B or human papillomavirus E6 systems. Thus, the 45. Swenson, J. J., Mauser, A. E., Kaufmann, W. K., and Kenney, S. C. (1999) main blocking mechanisms of checkpoint signaling by EBV J. Virol. 73, 6540–6550 would be through regulation of p53. It remains to be clarified 46. Lin, W. C., Lin, F. T., and Nevins, J. R. (2001) Genes Dev. 15, 1833–1844 47. Stevens, C., Smith, L., and La Thangue, N. B. (2003) Nat. Cell Biol. 5, 401–409 whether degradation of p53 is dependent on its binding to the 48. Hammerschmidt, W., and Sugden, B. (1988) Cell 55, 427–433 BZLF1 protein. 49. Tsurumi, T. (2001) Curr. Top. Microbiol. Immunol. 258, 65–87 50. Bell, P., Lieberman, P. M., and Maul, G. G. (2000) J. Virol. 74, 11800–11810 REFERENCES 51. Querido, E., Blanchette, P., Yan, Q., Kamura, T., Morrison, M., Boivin, D., Kaelin, W. G., Conaway, R. C., Conaway, J. W., and Branton, P. E. (2001) 1. Westphal, C. H. (1997) Curr. Biol. 7, R789–R792 2. Shiloh, Y. (2003) Nat. Rev. Cancer 3, 155–168 Genes Dev. 15, 3104–3117 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment *

Download PDF
8 pages

Loading next page...
 
/lp/american-society-for-biochemistry-and-molecular-biology/epstein-barr-virus-lytic-replication-elicits-atm-checkpoint-signal-kKUI0xRdaV

References (55)

Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2005 Elsevier Inc.
ISSN
0021-9258
eISSN
1083-351X
DOI
10.1074/jbc.m411405200
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 9, Issue of March 4, pp. 8156–8163, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment* Received for publication, October 6, 2004, and in revised form, December 15, 2004 Published, JBC Papers in Press, December 15, 2004, DOI 10.1074/jbc.M411405200 Ayumi Kudoh‡§ , Masatoshi Fujita‡ , Lumin Zhang‡§, Noriko Shirata‡, Tohru Daikoku‡, Yutaka Sugaya‡, Hiroki Isomura‡, Yukihiro Nishiyama§, and Tatsuya Tsurumi‡** From the ‡Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan, the Virology Division, National Cancer Center, Chuo-ku, Tokyo 104-0045, Japan, and the §Department of Virology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan related (ATR), respond to a variety of abnormal DNA structures When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or ar- and initiate signaling cascades leading to a DNA damage check- rest of cell-cycle progression, providing time for DNA point (1). ATM responds to the presence of DNA double-strand repair. Induction of the Epstein-Barr virus (EBV) lytic breaks (DSBs) induced by ionizing radiation (2). On the other program elicited a cellular DNA damage response, with hand, the ATR pathway can be stimulated by hydroxyurea, UV activation of the ataxia telangiectasia-mutated (ATM) light, and base-damaging agents that interfere with the move- signal transduction pathway. Activation of the ATM- ment of replication forks (3). The ATR pathway also responds Rad3-related (ATR) replication checkpoint pathway, in to DSBs, but more slowly than ATM (4). contrast, was minimal. The DNA damage sensor Mre11- A variety of checkpoint proteins have been identified as Rad50-Nbs1 (MRN) complex and phosphorylated ATM substrates for ATM and ATR kinases, including the checkpoint were recruited and retained in viral replication com- kinases Chk1 and Chk2, as well as H2AX (5) and p53 (2, 6). partments, recognizing newly synthesized viral DNAs as ATM phosphorylates Chk2 at several sites including Thr-68, abnormal DNA structures. Phosphorylated p53 also be- followed by Chk2 activation (6–9). Chk1 is phosphorylated at came concentrated in replication compartments and Ser-345 by ATR in response to UV and hydroxyurea, leading to physically interacted with viral BZLF1 protein. Despite a 3–5-fold increase in Chk1 activity (6, 7, 10). ATM is activated the activation of ATM checkpoint signaling, p53-down- by intermolecular autophosphorylation on Ser-1981 (11). Both stream signaling was blocked, with rather high S-phase Mre11 and Nbs1 are also targets of ATM and possibly ATR (10, CDK activity associated with progression of lytic infec- tion. Therefore, although host cells activate ATM check- 12–14). The MRN complex consisting of Mre11, Rad50, and point signaling with response to the lytic viral DNA Nbs1 has been proposed to facilitate ATM activation (15–17) synthesis, the virus can skillfully evade this host check- and recently demonstrated to function upstream of ATM acti- point security system and actively promote an S-phase- vation as a damage sensor, in addition to acting as an effector like environment advantageous for viral lytic replication. of ATM signaling (15, 18). ATM/ATR-initiated checkpoint signaling induces p53- dependent and p53-independent responses. The p53-dependent Eukaryotic cells exhibit a variety of physiological responses, cell cycle checkpoint features p21-mediated inactivation of including cell cycle arrest, activation of DNA repair and apo- Cdk2/cyclin E (19–21), while Chk2 inhibits Cdk2/cyclin E ac- ptosis, upon DNA damage. Sets of checkpoint proteins that tivity by phosphorylation of Cdk2 at Tyr-15, via down-regula- have been conserved with evolution are rapidly induced to tion of CDC25A phosphatase (4), in a p53-independent fashion. prevent replication or segregation of damaged DNA before re- Among Cdk2-targets, the Rb protein is most important for cell pair is completed. The related phosphatidylinositol 3-like ki- cycle progression and the checkpoint pathways result in its nases, ataxia telangiectasia-mutated (ATM) and ATM-Rad3- hypophosphorylation, leading to G or G /M cell cycle arrest. 1 2 The Epstein-Barr virus (EBV) is a human herpes virus that infects 90% of individuals. Primary EBV infection targets rest- * This work was supported by Grants-in-aid for Scientific Research ing B lymphocytes, inducing their continuous proliferation. In on Priority Areas from the Ministry of Education, Science, Sports, the B lymphoblastoid cell lines (LCL) only limited numbers of Culture and Technology of Japan (16017322 and 15390153, to T. T.). The costs of publication of this article were defrayed in part by the viral genes are usually expressed and there is no production of payment of page charges. This article must therefore be hereby marked virus particles, this being called latent infection. In the latent “advertisement” in accordance with 18 U.S.C. Section 1734 solely to state, EBV maintains its 170 kbp genome as complete, multiple indicate this fact. copies of plasmids that are synthesized only once in each S- ¶ Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. phase by the host cell replication machinery, following the ** To whom correspondence should be addressed: Division of Virol- rules of chromosome replication (22). ogy, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa- EBV-infected cell lines usually contain a small subpopula- ku, Nagoya 464-8681, Japan. Tel./Fax: 81-52-764-2979; E-mail: tion of cells that have switched spontaneously from a latent [email protected]. The abbreviations used are: ATM, ataxia telangiectasia-mutated; stage of infection into the lytic cycle. The mechanism of switch- EBV, Epstein-Barr virus; PBS, phosphate-buffered saline; h.p.i., h post- ing is not fully understood, but one of the first detectable induction; Gy, Gray; ATR, ATM-Rad3-related; DSB, double-stranded changes is expression of the BZLF1 immediate-early gene prod- break; FISH, fluorescence in situ hybridization; BrdUrd, bromode- uct. The BZLF1 protein, together with the other immediate- oxyuridine; MRN, Mre11-Rad50-Nbs1; mCSK butter, modified cy- toskelton butter; Pipes, 1,4-piperazinediethanesulfonic acid. early protein, BRLF1 protein, transactivates viral promoters 8156 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. DNA Damage Signaling during EBV Lytic Replication 8157 molecular mass proteins (180 kDa) or phosphorylated form of Rb pro- (23) and leads to an ordered cascade of viral early and late gene teins, gradient SDS-PAGE or SDS-7.5% PAGE (acrylamide, 72; bisacryl- expression. Early gene products include proteins involved in amide, 1), respectively, were applied. The proteins were then processed as viral DNA replication and DNA metabolism. The lytic phase of described previously (26). Detection of target proteins was with an en- EBV DNA replication is dependent on seven viral replication hanced chemiluminescence detection system (Amersham Biosciences). proteins: BZLF1, an oriLyt-binding protein; BALF5, a DNA Immunofluorescence—Cells were treated with 0.5% Triton X-100- polymerase; BMRF1, a polymerase processivity factor; BALF2, mCSK buffer (10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl ,1mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl a single-stranded DNA-binding protein; and BBLF4, BSLF1, 2 fluoride, 10 g/ml aprotinin, and 0.5% Triton X-100) for 10 min on ice, and BBLF2/3, predicted to be helicase-, primase-, and helicase followed by fixation with 70% methanol for 20 min on ice. The fixed cells primase-associated proteins, respectively (24). Viral lytic replica- were washed with PBS and blocked for 20 min in 10% normal goat tion occurs in discrete sites in nuclei, called replication compart- serum in PBS. Staining with a mouse monoclonal antibody to phospho- ments in which viral replication proteins are assembled (25). rylated Ser-1981 of ATM, mouse monoclonal antibodies to NBS1, We have previously demonstrated that induction of the EBV 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% lytic program results in inhibition of replication of cellular goat serum. Staining with a rabbit polyclonal antibody to BALF2, a DNA as well as explosive replication of viral DNA (26). The mouse monoclonal antibody to BMRF1 and a rabbit polyclonal antibody levels of p53 and CDK inhibitors remain unchanged through- to BZLF1 was carried out for1hat room temperature. Species-specific out the lytic infection, while the amounts of cyclin E/A and the secondary antibodies were applied for1hat room temperature. Alexa- hyperphosphorylated form of Rb increase as lytic infection 488 and Alexa-594, highly cross-absorbed secondary reagents, were progresses. The resultant S-phase-like cellular condition is purchased from Molecular Probes and slides were mounted in Vecta- shield (Vector Labs) and analyzed by fluorescence confocal microscopy. found to be essential for the transcription of viral immediate- Confocal fluorescence images were captured and processed using a early and early genes probably attributed to transcription fac- Radiance2000 Confocal System (Bio-Rad). All the primary antibodies tors such as E2F-1 and Sp1 expressed during S phase (27). were employed at 1:100 dilutions, and the secondary antibodies at 1:500 It is of interest to determine whether host cells can monitor dilutions. All washes after antibody incubations were performed with EBV lytic replication as DNA damage or abnormal DNA and, if 0.05% Tween-20 in PBS at room temperature. The specificity of the so, how EBV blocks the checkpoint signaling to avoid G or second antibodies and reliability of discrimination with fluorescent microscopy filters were tested. When cells were stained singly for either G /M cell cycle arrest and apoptosis. We have previously iso- antigen with inappropriate combinations of first and second antibodies, lated EBV latently infected Tet-BZLF1/B95-8 cells in which no fluorescence was observed and also no immunofluorescence was exogenous BZLF1 protein is conditionally expressed under the observed with alternate filters. control of a tetracycline-regulated promoter (26). Using this For staining the BrdUrd-incorporated DNA, cells were treated for 10 system, we show here for the first time that induction of EBV min with 2 N HCl containing 0.5% Triton X-100 to expose the incorpo- lytic replication elicits a cellular DNA damage response de- rated BrdUrd residues before blocking. The cells were washed and neutralized with 0.1 M sodium tetraborate (pH 9.0) for 5 min of incu- pendent on ATM. DNA damage sensor MRN complex and phos- bation. For BrdUrd staining, Alexa Fluor 488-conjugated anti-BrdUrd phorylated ATM are recruited to viral replication compart- mouse monoclonal antibody was used. ments, presumably recognizing newly synthesized viral DNAs Fluorescence in Situ Hybridization (FISH)—EBV BamHI-W frag- as abnormal DNA structures. However, the ATM checkpoint ment was labeled with Chroma Tide Alexa Fluor 594-5-dUTP (Molec- signaling was blocked at downstream of p53. Therefore, al- ular Probes, Inc.) and used for the detection of amplified EBV genomes. though EBV lytic replication elicits ATM-dependent DNA dam- At first, immunostaining was performed as described above and then refixed in 4% paraformaldehyde to cross-link bound antibodies. After age response, the virus can skillfully block the host response permeabilizing in 0.2% Triton X-100 (20 min on ice), cells were digested and actively promote an S-phase-like environment advanta- with RNase A, dehydrated in ethanol, air-dried, and immediately cov- geous for viral lytic replication. ered with the probe mixture containing 50% formamide in 2 SSC containing probe DNA (10 ng/l), 10% dextran sulfate, salmon sperm EXPERIMENTAL PROCEDURES DNA (0.1 g/l), and yeast tRNA (1 g/l). Probe and cells were simul- Cells—Tet-BZLF1/B95-8 cells, a marmoset B-cell line latently in- taneously heated at 94 °C for 4 min and incubated overnight at 37 °C. fected with EBV (26), and Tet-BZLF1/Akata cells, human EBV-positive After hybridization, specimens were washed at 37 °C with 50% form- Burkitt’s lymphoma cells (27), were maintained in RPMI medium sup- amide in 2 SSC (two times for 15 min each) and 2 SSC. Finally, cells plemented with 1 g/ml of puromycin, 250 g/ml of hygromycin B, and were equilibrated in PBS and mounted in vectashield (Vector Labora- 10% tetracycline-free fetal calf serum. To induce lytic EBV replication, tories, Inc.). a tetracycline derivative, doxycycline, was added to the culture medium Immunoprecipitation—Cells were lysed in 1 ml of EBC lysis buffer at a final concentration of 2 g/ml. B95-8 cells were cultured in RPMI (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40) containing medium supplemented with 10% fetal calf serum. 100 mM sodium fluoride, 2 mM sodium vanadate, and protease inhibitor Antibodies—Primary antibodies were purchased from Cell Signaling mixture (Sigma; 25 l/ml), and then sonicated. The lysates were cen- (ATM-S1981, Chk1, Chk1-S345, Chk2-T68, p95/NBS1-S343, and p53- trifuged at 18,000  g for 20 min at 4 °C, and immunoprecipitation was S15), Genetex (ATM-2C1), BD Transduction Laboratories (Cdk1, Cdk2, performed using 1 mg of the supernatant and 5 g of anti-BZLF1 Chk2, Mre11, and Nbs1), Oncogene (p53, p21 and MDM2), Santa Cruz protein-specific IgG-beads or control rabbit IgG beads, with gentle Biotechnology (cyclin A-C19, cyclin B1-GCN1, cyclin E -M20, and E2F1- rocking for1hat4 °C. Ternary protein A-antibody-antigen complexes KH95), Upstate (H2AX-S139), Chemicon (EBV BMRF1-R3) and Argene were collected by centrifugation and washed three times with NET-gel (EBV BRLF1–8C12). Rabbit polyclonal antibodies to BZLF1, BALF2, buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Nonidet P-40, and BBLF2/3, and BALF5 proteins were prepared as described (26). Highly 1mM EDTA). The immunoprecipitates were subjected to SDS- cross-absorbed secondary reagents for dual-color detection (Alexa-488 10%PAGE followed by immunoblotting analyses. and 594) were from Molecular Probes. In Vitro Protein Kinase Assay—Cells were washed with ice-cold PBS Protein Preparation—Tet-BZLF1/B95-8, Tet-BZLF1/Akata, and and then lysed with EBC lysis buffer containing 100 mM sodium fluo- B95-8 cells were harvested at the indicated times post-treatment with ride, 2 mM sodium vanadate, and multiple protease inhibitors (Sigma; doxycycline, washed with phosphate-buffered saline (PBS), and treated 25 l/ml) for 5 min on ice followed by centrifugation at 18,000  g for 20 with lysis buffer (0.02% SDS, 0.5% Triton X-100, 300 mM NaCl, 20 mM min at 4 °C. For kinase assays 200 g of aliquots of protein extracts Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol) for 20 min on ice. were incubated with 1 g of each antibody in a final volume of 500 l for Multiple protease inhibitors (Sigma; 25l/ml), 200 M sodium vana- 1 h at 4 °C to precipitate cyclin-CDK complexes: C-19 against cyclin A, date, and 20 mM sodium fluoride were added to the buffer. Samples M-20 against cyclin E and GNS1 against cyclin B. Immune complexes were centrifuged at 18,000  g for 10 min at 4 °C, and clarified cell absorbed onto protein A-Sepharose were washed twice with ice-cold extracts were assayed for protein concentration using a Bio-Rad kit. NET-gel buffer, and then once with basic kinase buffer (200 mM Tris- Immunoblot Analysis—Equal amounts of proteins (2050 g) were HCl, pH 7.5, 10 mM MgCl ,1mM EDTA, 1 mM dithiothreitol). The loaded into each lane for SDS-10% polyacrylamide (acrylamide, 29.2; immunoprecipitates were resuspended in 24 l of kinase buffer (200 mM bisacrylamide, 0.8) gel electrophoresis (SDS-PAGE). To separate high Tris-HCl, pH 7.5, 10 mM MgCl ,1mM EDTA, 1 mM dithiothreitol, 50 M 2 8158 DNA Damage Signaling during EBV Lytic Replication ATP containing 5 Ci of [- P]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 lof5 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 g/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  10 cells and quantified. Dot-blot hybridization was performed, and quantification of the copy numbers of viral genome per cell were determined as described previously (26). RESULTS 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 pro- teins after induction of lytic replication with doxycycline were described previously (26). BZLF1 protein became detectable 4 h post-induction (h.p.i.) (26) and reached a plateau at 48 h.p.i. (Fig. 1A). The other immediate-early protein, the BRLF1 pro- tein also appeared at 6 h.p.i (26) 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-associ- ated 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 path- way 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 (11). 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 phos- phorylate Thr-68 on Chk2, which is required for its activation (9, 28). Immunoblotting with anti-Chk2 Thr-68-specific anti- body 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 (29, 30), was also exam- ined. 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 FIG.1. Activation of DNA damage responsive proteins upon expression levels of p53 protein throughout lytic infection, in induction of lytic replication. Tet-BZLF1/B95-8 cells or B95-8 cells agreement with our previous observation (26). were cultured in the presence of 2 g/ml of doxycycline, harvested at the indicated times. Equal amounts of proteins for each sample (20  50 g) It should be noted that not only activated ATM but also ATR were applied for immunoblot analyses. A, expression profile of EBV kinases phosphorylate Chk2 kinase at Thr-68 and up-regulate lytic proteins in lytic program-induced Tet-BZLF1/B95-8 cells. B, ex- its activity (9, 28). Phosphorylation of histone H2AX or p53 at pression levels of ATM autophosphorylated at Ser-1981, ATM, Chk2 Ser-15 is also carried out by ATM/ATR kinases (31, 32). There- phosphorylated at Thr-68, Chk2, Chk1 phosphorylated at Ser-345, Chk1, H2AX phosphorylated at Ser-139 (H2AX), p53 phosphorylated fore, EBV lytic replication could activate the ATM, ATR, or at Ser-15, and p53, were determined by immunoblotting. C, the Tet- both kinases. The ATR kinase responds primarily to DNA BZLF1/Akata cells treated with 2 g/ml of doxycycline were harvested replication stress during S phase (3). It can also respond to at indicated times. Clarified cell lysates were prepared and applied for DSBs if within the S phase, but less efficiently than ATM. In Western blot analyses with each antibody as indicated. D, the Tet- BZLF1/Hela cells treated with 2 g/ml of doxycycline were harvested at contrast to Chk2, phosphorylation of Chk1 at Ser-345 is known indicated times. Clarified cell lysates were prepared and applied for to be carried out mainly by ATR kinase, leading to its activa- Western blot analyses with each antibody as indicated. HU, cells were tion (10). Therefore, we examined Chk1 phosphorylation at treated with 5 mM of hydroxyurea for 12 h and harvested. IR, cells were Ser-345 in lytic replication-induced B95-8 cells, but no signifi- exposed to -radiation with 10 Gy and harvested 4 h post-treatment. cant phosphorylation was observed (Fig. 1B). Treatment of cells with hydroxyurea, a well-studied activator of the replica- Thus, we conclude that EBV lytic replication elicits activation tion checkpoint, clearly induced Chk1 phosphorylation (Fig. of ATM DNA damage checkpoint signaling rather than the 1B), showing the ATR/Chk1 pathway to be intact in the cells. ATR pathway that responds to replication stress. DNA Damage Signaling during EBV Lytic Replication 8159 FIG.2. Subcellular localization of ATM Ser-1981 after induc- tion of lytic replication. A, architecture of viral replication compart- ments. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 g/ml doxycycline for 14 h and newly synthesized DNAs were labeled with 10 M BrdUrd for 1 h prior to harvest. Cells were treated with 0.5% Triton FIG.3. NBS1 and Mre11 proteins constituting the MRN com- X-100-mCSK buffer and were then fixed with methanol viral replication plex are recruited to EBV replication compartments. A, Tet- proteins (BMRF1 and BALF2 proteins), newly synthesized DNA, or BZLF1/B95-8 cells were cultured in the presence of 2 g/ml of doxycy- viral DNA was visualized by immunofluorescence, BrdUrd staining, or cline for the indicated times, and expression levels of Nbs1 and Mre11 FISH analyses, respectively. Right panels are merged images. B, sub- were analyzed by immunoblotting with each antibody as indicated. B, nuclear localization of phosphorylated ATM. Tet-BZLF1/B95-8 cells recruitment of Nbs1 and Mre11 constituting the MRN complex to viral were cultured in the absence (panel a) or presence of 2 g/ml of doxy- replication compartments. Tet-BZLF1/B95-8 cells were cultured in the cycline and harvested at 14 h.p.i. (panels b and d) or 24 h.p.i. (panels c absence (panels a, b, and d) or presence (panels c and e)of2 gof and e). Cells were treated with 0.5% Triton X-100-mCSK buffer. The doxycycline/ml and harvested at 14 h.p.i. Cells were treated with 0.5% nonionic-detergent-extracted cells were then fixed with methanol. Pro- Triton X-100-mCSK buffer, fixed with methanol and coimmunostained teins (BALF2 protein and ATM phosphorylated at Ser-1981) and viral with antibodies to BALF2 protein and Nbs1 protein (panels a, b, and c) DNA was visualized by immunofluorescence and FISH analyses, re- or Mre11 protein (panels d and e). Panel b, Tet-BZLF1/B95-8 cells were spectively. Right panels are merged images. exposed to 10 Gy  irradiation and harvested 4 h post-treatment. Cells were processed in parallel with the lytic program-induced cells. Right panels are merged images. To ascertain whether lytic replication elicits ATM/Chk2 DNA damage checkpoint signaling in other EBV-latently in- fected B cells, we examined Akata cells, an EBV-positive B cell thesis, these were used as markers for viral replication com- line derived from a Burkitt’s lymphoma. As shown in Fig. 1C, partments. We examined whether activated DNA damage re- phosphorylation of ATM and Chk2 was again observed upon sponsive proteins accumulate in such foci after induction of induction of lytic replication. Furthermore, as shown in Fig. lytic infection. As shown in Fig. 2B, in the lytic replication- 1D, it should be noted that expression of the BZLF1 protein induced cells, ATM phosphorylated at Ser-1981 was found to be alone in Hela cells did not phosphorylate NBS1 Ser-343, Chk2 resistant to detergent extraction and became colocalized with at Thr-68, and p53 at Ser-15 as judged by Western blotting, viral DNAs in the replication compartments, strongly suggest- suggesting that expression of the BZLF1 protein itself cannot ing that activated ATM recognizes and binds to newly synthe- elicit DNA damage response checkpoint signaling pathways. sized viral DNA. In contrast, phosphorylated ATM was not Phosphorylated ATM Accumulates in Viral Replication Com- observed in the latently infected cells (Fig. 2B, panel a). partments After Induction of Lytic Replication—Many proteins The Nbs1 and Mre11 Proteins Constituting the MRN Com- involved in the DNA damage response accumulate in foci at plex Accumulate in Viral Replication Foci—The MRN complex consisting of Mre11, Rad50, and Nbs1 has been suggested to sites of DSBs or abnormal DNA structures such as single- stranded DNA (33). It has previously been demonstrated that act as a damage sensor (14, 18), facilitating ATM activation (15). After ionizing radiation, relocalization of the MRN com- ATM protein becomes associated with chromatin upon ionizing radiation, using a biochemical fractionation procedure in which plex at sites of damage is readily observed in detergent-ex- tracted cells (36) and independent of ATM and a portion of the ATM pool was found to be resistant to detergent H2AX (18, 31, extraction after treatment with agents that cause DSBs (18, 36). As shown in Fig. 3A, the levels of Mre11 and Nbs1 were 34). EBV lytic DNA replication occurs at discrete sites in nu- constant throughout the lytic infection, unlike the case for clei, called replication compartments, where viral replication adenovirus infection which degrades the MRN complex (18, proteins cluster and viral DNAs are synthesized (25). Lytic 37). Activated ATM also phosphorylates Ser-343 on Nbs1 (12, replication-induced Tet-BZLF1/B95-8 cells were extracted with 13). As shown in Fig. 3A, increase in levels of phosphorylated 0.5%Triton X-100-mCSK buffer to solubilize DNA-unbound form of Nbs1 at Ser-343 was observed, this becoming detectable forms of viral or cellular proteins (35). As shown in Fig. 2A, the after12 h.p.i. Next, we assessed the effect of induction of EBV BMRF1 and BALF2 viral replication proteins were colocalized lytic replication on the localization of Mre11 and Nbs1 by in the nuclei after induction of lytic replication. The sites were immunofluorescence after detergent extraction (Fig. 3B). No completely coincided with the localized foci of newly synthe- staining with Mre11-specific or Nbs1-specific antibodies was sized viral DNA as judged by BrdUrd incorporation and FISH observed in the detergent-treated latently infected cells (Fig. analyses (Fig. 2A, panels b and c). Thus, since the BALF2 or 3B, panels a and d). As shown in Fig. 3B, panel b, ionizing BMRF1 protein-localized sites represent loci of viral DNA syn- radiation resulted in distinct staining of Nbs1 in the nuclei, 8160 DNA Damage Signaling during EBV Lytic Replication FIG.4. S-phase-like environment after induction of lytic replication. Tet-BZLF1/B95-8 or B95-8 cells were cultured in the presence of 2 g/ml of doxycycline and harvested at the indicated times. Clarified cell lysates were prepared, and the proteins were analyzed by Western blotting cip1/waf1 with specific antibodies. A, expression levels of p21 and MDM2 after treatment with doxycycline in Tet-BZLF1/B95-8 cells and B95-8 cells. IR, Tet-BZLF1/B95-8 cells were exposed to 10 Gy  irradiation and harvested 4 h post-treatment. B, acceleration of proteasomal degradation of p53 in lytic phase of EBV replication. Tet-BZLF1/B95-8 cells were cultured in the presence (Dox ) or absence (Dox )of2 g/ml of doxycycline. Proteasome inhibitor, 20 M MG132 plus 0.1% Me SO (MG132), or 0.1% Me SO as control (MG132) was added to the culture at 16 h.p.i., and 2 2 cells were harvested at 24 h.p.i. C, accumulation of E2F-1 accompanied by hyperphosphorylation of Rb after induction of the lytic program in Tet-BZLF1/B95-8 cells. The proteins were separated by SDS-7.5% PAGE (for RB) or SDS-10% PAGE (for others) and analyzed by Western blotting with antibodies as indicated. The slower migrating bands are hyperphosphorylated forms of the Rb protein (ppRB and pRB). The faster migrating band is the hypophosphorylated form of the Rb protein (RB). D, elevation of cyclin A- or cyclin E-associated CDK activity during EBV lytic replication. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 g/ml of doxycycline and harvested at the indicated times. Equal amounts of whole cell extracts were prepared and used for immunoprecipitation (IP) with antibodies against each of the indicated cyclins, and then kinase assays were performed using histone H1 as substrate, as described under “Experimental Procedures.” Lower panel shows the densitometric analysis of cyclin A-, cyclin E-, and cyclin B-associated CDK activities. The signal intensity was quantified with an Image Guider (BAS2500, Fuji film). indicating that the MRN complex is activated and retained in throughout lytic replication (Fig. 1B). Therefore, we exam- the damaged sites. Upon induction of lytic replication, Mre11 ined expression levels of p53-transcriptional targets such as cip1/waf1 and Nbs1 proteins became resistant to detergent treatment p21 , a cyclin-dependent kinase inhibitory protein, and and colocalized predominantly in viral replication compart- MDM2, a ubiquitin protein ligase, for p53 (Fig. 4A). When cip1/waf1 ments represented by the BALF2 staining (Fig. 3B, panels c Tet-BZLF1/B95-8 cells were irradiated, the levels of p21 and e). Once the pools of endogenous Mre11 and Nbs1 protein and MDM2 were elevated remarkably in response. In contrast, were concentrated in this way, the associated fluorescence be- the level of MDM2 ubiquitin ligase came down with progres- cip1/waf1 came resistant to extraction with a mild detergent-containing sion of lytic replication, and the level of p21 was low and extraction buffer, indicating that the Mre11 and Nbs1 proteins almost unchanged throughout the lytic replication. These sug- became not only redistributed to, but also retained within the gest that EBV lytic program possesses a defense system to close vicinity of newly synthesized viral DNA. It is likely that prevent p53 downstream signaling. The levels of p53-independ- kip1 ink4a the MRN complex recognizes newly synthesized viral genomic ent CDK inhibitors such as p27 and p16 were also DNA in the replication compartments as abnormal DNA struc- unchanged (data not shown). These observations suggest that tures and binds to them. p53 downstream signaling is blocked during EBV lytic infection Inhibition of p53 Downstream Target Gene Expression after despite the appearance of phosphorylated p53. Induction of EBV Lytic Infection—As described above, induc- Next, the relative abundance of p53 was analyzed by West- tion of EBV lytic replication elicited phosphorylation of ATM, ern blot in the presence of the chemical proteasome inhibitor, Nbs1, H2AX, Chk2, and p53. Phosphorylation of p53 at Ser-15 MG132. As shown in Fig. 4B, the amounts of p53 in EBV in response to DNA damage usually correlates with both accu- latently infected cells were almost constant in the absence and mulation of total p53 protein as well as with the ability of p53 presence of MG132. By contrast, MG132 stabilized p53, result- to transactivate downstream target genes in wild-type cells ing in comparative accumulation of p53 in the lytic infection- (38). However, the level of p53 here proved to be constant induced cells. Thus, it was clearly demonstrated that p53 turn- DNA Damage Signaling during EBV Lytic Replication 8161 over is regulated by proteasome degradation after induction of the lytic infection. These results are intriguing because the degradation of p53 via proteasome appears to be one of the mechanisms that explain why the phosphorylated form of p53 at Ser-15 cannot transactivate its downstream factors such as p21 and MDM2. Since the level of MDM2 ubiquitin ligase came down with progression of lytic replication, degradation of p53 might be mediated by a direct interaction and recruitment of ubiquitin ligase activity other than MDM2. Activation of S-phase-promoting CDK Activity throughout Lytic Infection—As shown in Fig. 4C, we observed that the levels of cyclin E and cyclin A. continued to be elevated, whereas the level of cyclin B was constant during the lytic replication, con- firming our previous report (26). These data strongly suggest that S-phase-promoting CDK, namely cyclin A/E-Cdk2, is acti- vated during lytic infection. In fact, as shown in Fig. 4D, cyclin E- and cyclin A-associated CDK activity increased as lytic replica- tion progressed, whereas cyclin B-associated kinase activity was unchanged and rather down-regulated at 48 h.p.i. Slow migrat- ing hyperphosphorylated Rb proteins accumulated with progres- sion of lytic infection (Fig. 4C), probably because of elevated cyclin E- and A-associated CDK activity. Also, the levels of E2F-1 increased with progression of lytic infection (Fig. 4C). Hyperphos- phorylation of Rb may result in release of an active form of E2F-1, which binds to its own binding site on E2F-1 promoters and enhances expression (39). Thus, these observations clearly indi- cate that EBV lytic replication occurs in an S phase-like cellular environment regardless of elicitation of the ATM DNA damage response. p53 Physically Interacts with EBV BZLF1 Protein in Vivo and Is Recruited to Viral Replication Compartments—Zhang et al. (40) has previously reported that the BZLF1 protein inhibits transactivation activity of p53, likely through the direct inter- action demonstrated using an adenovirus overexpression sys- FIG.5. Phosphorylated p53 interacts with the BZLF1 protein. tem. To confirm actual protein-protein interaction in vivo and A, immunoprecipitation of the BZLF1 protein with an anti-p53 specific resolve the paradox between induction of ATM DNA damage antibody. Tet-BZLF1/B95-8 cells were untreated or treated with 2 g/ml doxycycline and harvested at 48 h.p.i. Clarified lysates were response and inhibition of p53 downstream events, immuno- prepared and subjected to immunoprecipitation (IP) analysis with anti- precipitation analyses with an anti-BZLF1 protein-specific an- BZLF1 IgG or control rabbit IgG beads. Aliquots of the immunoprecipi- tibody were performed with lytic replication-induced B95-8 cell tated proteins and lysates (Input) were analyzed by Western blotting extracts. As shown in Fig. 5A, the antibody immunoprecipi- with anti-p53 monoclonal IgG, anti-p53Ser-15 polyclonal IgG, or anti- BZLF1 polyclonal IgG. B, colocalization of p53 with the BZLF1 protein tated phosphorylated p53. Thus, the physical interaction be- in viral replication compartments. Tet-BZLF1/B95-8 cells were cultured tween the BZLF1protein and the phosphorylated form of p53 in the absence (panels a and c) or presence (panels b and d)of2 gof was confirmed not only in the overexpression system (40) but doxycycline/ml and harvested at 24 h.p.i. Cells were treated with 0.5% also in the lytic phase-induced B95-8 cells. Triton X-100-mCSK buffer, fixed with methanol, and coimmunostained with the indicated antibodies. Panels a and b show images of BZLF1 Next, we examined the localization of p53 using a specific protein and p53. Panels c and d show images of BMRF1 protein and p53 antibody to p53 or phospho-Ser-15 p53. In latently infected phosphorylated at Ser-15. Right panels are merged images. cells p53 was sensitive to detergent treatment and no staining with a p53-specific antibody was observed (Fig. 5B, panels a and c). In contrast, the p53-associated fluorescence became phosphorylation of target proteins of ATM kinase activity such resistant to extraction by a mild detergent-containing buffer as Chk2 at Thr-68, ATM at Ser-1981, and Nbs1 at Ser-343, the after induction of lytic replication. Thus, p53 became re- expression levels of viral replication proteins were not affected tained in the nuclei and colocalized with the BZLF1 protein at all (Fig. 6A). These data suggest that a caffeine-sensitive in the replication compartments. Also, p53 phosphorylated at kinase is involved in the checkpoint signaling evoked during Ser-15 was recruited to viral replication compartments. the EBV lytic infection, while ATM signaling is not absolutely Taken together, the results clearly demonstrated that phos- required for lytic replication. Moreover, in order to clarify the phorylated p53 was redistributed to viral replication com- effects of caffeine on EBV genome synthesis, Tet-BZLF1/B95-8 partments, associated with BZLF1 protein via physical cells were treated with doxycycline in the presence or absence interaction. of the compound. Total DNA was extracted from the cells, and Inhibition of ATM-dependent DNA Damage Response In- EBV DNA-specific signals were quantitated. As shown in Fig. duced by EBV Lytic Replication Does Not Affect Viral Lytic 6B, the copy number of the viral DNA was amplified up to more Replication—Phosphorylation states of ATM DNA damage re- than 1500 copies per cell after 48 h.p.i. in the absence of sponsive proteins and expression levels of viral lytic proteins caffeine. Inhibition by caffeine of ATM-dependent checkpoint during lytic infection with EBV were examined in the presence activation induced by EBV lytic replication did not affect viral or absence of caffeine, a dose that inhibits the kinase activity of lytic replication at all. It is likely that since ATM DNA damage both ATM and ATR in vitro (41). Although caffeine treatment signaling induced by the lytic replication is blocked at least at of lytic program-induced Tet-BZLF1/B95-8 cells abrogated the level of p53 by the BZLF1 protein, inhibition of ATM/ATR 8162 DNA Damage Signaling during EBV Lytic Replication FIG.6. Inhibition by caffeine of ATM DNA damage checkpoint activa- tion induced by the lytic replication. A, lytic replication was induced in Tet- BZLF1/B95-8 cells with doxycycline in the presence or absence of 5 mM caffeine and harvested at indicated times. In parallel, uninduced Tet-BZLF1/B95-8 cells were also cultured in the presence of caffeine as a control. Clarified cell lysates were pre- pared, and the proteins were separated by SDS-10% PAGE and analyzed by Western blotting with specific antibodies. B, Tet- BZLF1/B95-8 cells were cultured with doxycycline (2 g/ml) in the presence (E) or absence (●)of5mM caffeine. Cells were harvested at the indicated times, and vi- ral DNA synthesis was determined by slot blot assay as described under “Experi- mental Procedures.” kinase activity by caffeine might have almost no effect on viral phorylation of Ser-345 on Chk1 (Fig. 1B). Further, recruitment lytic replication. We conclude that ATM signaling is not abso- of ATR to EBV replication compartments as judged by confocal lutely required for lytic replication. immunostaining analyses was not observed (data not shown). The ATR kinase responds primarily to chromosomal DNA rep- DISCUSSION lication stress during S phase (3). Taken together, our results It has been clearly demonstrated here that EBV lytic repli- indicate that EBV lytic replication preferentially activates the cation indeed induced the ATM DNA damage response, which ATM DNA damage response. At 24 h after lytic induction, some was blocked through the interaction between the BZLF1 pro- cells exhibited BrdUrd staining throughout nuclei. We could tein and p53. Also, activated Chk2 is known to inhibit Cdk2/ not observe any viral replication compartments stained with cyclin E or A activity by phosphorylation of Cdk2 at Tyr-15, via specific antibodies to the BMRF1 or the BALF2 lytic proteins in down-regulation of CDC25A phosphatase (4), in a p53-inde- such cells, suggesting that EBV lytic replication might not pendent fashion. Although Chk2 was phosphorylated by ATM occur in S-phase cells in which chromosomal DNA replication as shown in Fig. 1, cyclin A- and E-associated CDK2 activity has already started. If EBV lytic replication had arrested fork was increased with the progression of lytic replication. It has movements of chromosomal DNA replication, ATR DNA dam- been very recently reported that EBNA3a, one of viral proteins age checkpoint could be activated. Further experiments are expressed during latent infection, interacts with Chk2 to dis- needed to clarify this point. rupt G2/M checkpoint (42), suggesting inhibition of p53-inde- The expression level of E2F-1 is elevated as lytic replication pendent DNA damage signaling pathway. Since the G -or progresses. Although the precise mechanism remains to be G /M-arrested state or apoptosis following DNA damage check- determined, maintaining low levels of CDK inhibitors during point signaling would be unsuitable for viral lytic replication, lytic infection might result in accumulation of the hyperphos- EBV would create the cell to become an S-phase-like condition. phorylated form of Rb protein by cyclin E and A associated ATM and the MRN complex function in a common pathway kinase activity. Phosphorylation of the Rb protein leads to (13), and the MRN complex can function to activate ATM as a release from its inhibitory effects on E2F-1 as transcription damage sensor, in addition to acting as an effector of ATM factor (43), thereby deregulating E2F-1 in favor of an S-phase signaling (18). Clustering of ATM, Mre11, and Nbs1 proteins to environment. E2F-1 can transactivate not only the cyclin E and the EBV replication compartments from early stages of lytic cyclin A genes but also its own expression. Alternatively, it has infection strongly suggests that these damage sensors recog- been very recently reported that the BZLF1 protein by itself nize newly synthesized viral DNAs as abnormal DNA struc- activates E2F-1, cyclin E, and Cdc25A involved in cell cycle tures. Although ATM/ATR kinase phosphorylates histone progression in telomerase-immortalized human keratinocytes H2AX and p53 at Ser-15, ATR kinase activity predominantly and primary tonsil keratinocytes (44) and further that the phosphorylates Chk1 at Ser-345 leading to increased Chk1 activity (6, 7, 10). Surprisingly, we could not detect any phos- other immediate-early transactivator, BRLF1 protein, can in- DNA Damage Signaling during EBV Lytic Replication 8163 3. Osborn, A. J., Elledge, S. J., and Zou, L. (2002) Trends Cell Biol. 12, 509–516 duce contact-inhibited, quiescent human fibroblasts to enter 4. Zhou, B. B., and Elledge, S. J. (2000) Nature 408, 433–439 the S phase with dramatic increase in the level of E2F-1 (45). 5. Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M., Also, activated ATM or Chk2 phosphorylate and activate E2F-1 and Bonner, W. M. (2000) Curr. Biol. 10, 886–895 6. Kastan, M. B., and Lim, D. S. (2000) Nat. Rev. Mol. Cell. Biol. 1, 179–186 in response to DNA damage (46, 47) and thus the fact that 7. Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas, J. (2001) Nature inhibition of ATM kinase activity by caffeine treatment re- 410, 842–847 8. Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K., and Elledge, S. J. duced the level of E2F-1 (data not shown) is in line with the (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10389–10394 literature. 9. Melchionna, R., Chen, X. B., Blasina, A., and McGowan, C. H. (2000) Nat. Cell Infection with an E4-deleted adenovirus results in synthesis Biol. 2, 762–765 10. Zhao, H., and Piwnica-Worms, H. (2001) Mol. Cell. Biol. 21, 4129–4139 of end-joined large viral genomes that are recognized as abnor- 11. Bakkenist, C. J., and Kastan, M. B. (2003) Nature 421, 499–506 mal DNA structures by the MRN complex, leading to activation 12. Gatei, M., Young, D., Cerosaletti, K. M., Desai-Mehta, A., Spring, K., Kozlov, of cell cycle checkpoints. As a result, ATM damage response S., Lavin, M. F., Gatti, R. A., Concannon, P., and Khanna, K. (2000) Nat. Genet. 25, 115–119 signaling is activated and Chk1, Chk2, 53BP1, p53, Nbs1 and 13. Wu, X., Ranganathan, V., Weisman, D. S., Heine, W. F., Ciccone, D. N., ATM are phosphorylated (18, 37). Similarly, in the case of EBV, O’Neill, T. B., Crick, K. E., Pierce, K. A., Lane, W. S., Rathbun, G., Living- intermediate replication products in lytic phase are thought to ston, D. M., and Weaver, D. T. (2000) Nature 405, 477–482 14. D’Amours, D., and Jackson, S. P. (2002) Nat. Rev. Mol. Cell. Biol. 3, 317–327 be large head-to-tail concatemers with branched structures 15. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L., and Shiloh, Y. that might be generated by homologous recombination coupled (2003) EMBO J. 22, 5612–5621 with replication events (48, 49). In homologous recombination 16. Usui, T., Ogawa, H., and Petrini, J. H. (2001) Mol. Cell 7, 1255–1266 17. Williams, B. R., Mirzoeva, O. K., Morgan, W. F., Lin, J., Dunnick, W., and repair the MRN complex, most likely with help of other nucle- Petrini, J. H. (2002) Curr. Biol. 12, 648–653 ase, might resects the DNA to provide ssDNA overhangs nec- 18. Carson, C. T., Schwartz, R. A., Stracker, T. H., Lilley, C. E., Lee, D. V., and essary for DNA pairing and strand exchange, since the MRN Weitzman, M. D. (2003) EMBO J. 22, 6610–6620 19. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, complex is known to possess exonuclease activity. The Rad50 J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell subunit of MRN has ATPase activity that is believed to facili- 75, 817–825 tate DNA unwinding. Thus, such higher order intermediate 20. Gu, Y., Turck, C. W., and Morgan, D. O. (1993) Nature 366, 707–710 21. Dulic, V., Kaufmann, W. K., Wilson, S. J., Tlsty, T. D., Lees, E., Harper, J. W., viral genome structures might be recognized and made by the Elledge, S. J., and Reed, S. I. (1994) Cell 76, 1013–1023 MRN complex. We speculate that this is the case and that the 22. Yates, J. L., and Guan, N. (1991) J. Virol. 65, 483–488 MRN complex then modifies the DNA to create a platform for 23. Flemington, E. K., Goldfeld, A. E., and Speck, S. H. (1991) J. Virol. 65, 7073–7077 ATM and other signaling factors (14). When viral lytic DNA 24. Fixman, E. D., Hayward, G. S., and Hayward, S. D. (1995) J. Virol. 69, replication is blocked by the addition of phosphonoacetic acid 2998–3006 (PAA), a herpes virus DNA polymerase specific inhibitor, viral 25. Takagi, S., Takada, K., and Sairenji, T. (1991) Virology 185, 309–315 26. Kudoh, A., Fujita, M., Kiyono, T., Kuzushima, K., Sugaya, Y., Izuta, S., prereplicative sites represented by staining of the BALF2 pro- Nishiyama, Y., and Tsurumi, T. (2003) J. Virol. 77, 851–861 tein are formed (50). Although viral lytic immediate-early and 27. Kudoh, A., Daikoku, T., Sugaya, Y., Isomura, H., Fujita, M., Kiyono, T., Nishiyama, Y., and Tsurumi, T. (2004) J. Virol. 78, 104–115 early proteins were expressed even in the presence of PAA, no 28. Ahn, J. Y., Schwarz, J. K., Piwnica-Worms, H., and Canman, C. E. (2000) accumulation of synthesized viral DNA, Nbs1, and phosphoryl- Cancer Res. 60, 5934–5936 ated ATM to the prereplicative sites was observed (data not 29. Yazdi, P. T., Wang, Y., Zhao, S., Patel, N., Lee, E. Y., and Qin, J. (2002) Genes shown). This result also supports the idea that newly synthe- Dev. 16, 571–582 30. Falck, J., Petrini, J. H., Williams, B. R., Lukas, J., and Bartek, J. (2002) Nat. sized viral DNA is recognized by cellular DNA damage sensors. Genet. 30, 290–294 During wild-type adenovirus infection, the E1b55k-E4orf6 31. Celeste, A., Fernandez-Capetillo, O., Kruhlak, M. J., Pilch, D. R., Staudt, D. W., Lee, A., Bonner, R. F., Bonner, W. M., and Nussenzweig, A. (2003) complex degrades the MRN complex, preventing signaling Nat. Cell Biol. 5, 675–679 through both ATM and ATR pathways (18, 37). Also, the com- 32. Tibbetts, R. S., Brumbaugh, K. M., Williams, J. M., Sarkaria, J. N., Cliby, plex could also function in the regulation of p53 levels by W. A., Shieh, S. Y., Taya, Y., Prives, C., and Abraham, R. T. (1999) Genes Dev. 13, 152–157 degradation of p53 through ubiquitination (51). Thus, adenovi- 33. Rouse, J., and Jackson, S. P. (2002) Science 297, 547–551 ruses appear to have evolved double-check mechanisms to 34. Andegeko, Y., Moyal, L., Mittelman, L., Tsarfaty, I., Shiloh, Y., and Rotman, G. block cell cycle checkpoint signaling pathways. Unlike the case (2001) J. Biol. Chem. 276, 38224–38230 35. Fujita, M., Ishimi, Y., Nakamura, H., Kiyono, T., and Tsurumi, T. (2002) with adenoviruses, lytic replication of EBV does not degrade J. Biol. Chem. 277, 10354–10361 the MRN complex since the levels of Mre11 and Nbs1 were 36. Mirzoeva, O. K., and Petrini, J. H. (2001) Mol. Cell. Biol. 21, 281–288 constant throughout the lytic replication (Fig. 3A). However, 37. Stracker, T. H., Carson, C. T., and Weitzman, M. D. (2002) Nature 418, 348–352 the BZLF1 protein binds to phosphorylated p53, preventing its 38. Siliciano, J. D., Canman, C. E., Taya, Y., Sakaguchi, K., Appella, E., and transcriptional activity (40). p53 turnover is also regulated by Kastan, M. B. (1997) Genes Dev. 11, 3471–3481 proteasome degradation even after induction of lytic infection 39. Johnson, D. G., Ohtani, K., and Nevins, J. R. (1994) Genes Dev. 8, 1514–1525 40. Zhang, Q., Gutsch, D., and Kenney, S. (1994) Mol. Cell. Biol. 14, 1929–1938 (Fig. 4B). As shown in Fig. 4A, the level of MDM2 ubiquitin 41. Sarkaria, J. N., Busby, E. C., Tibbetts, R. S., Roos, P., Taya, Y., Karnitz, L. M., ligase was reduced with progression of lytic replication. There- and Abraham, R. T. (1999) Cancer Res. 59, 4375–4382 fore, degradation of p53 might be mediated by direct interac- 42. Krauer, K. G., Burgess, A., Buck, M., Flanagan, J., Sculley, T. B., and Gabri- elli, B. (2004) Oncogene 23, 1342–1353 tion and recruitment of protein(s) with ubiquitin ligase activ- 43. Nevins, J. R. (1992) Science 258, 424–429 ity, besides MDM2. It is likely that unidentified viral protein(s) 44. Mauser, A., Holley-Guthrie, E., Zanation, A., Yarborough, W., Kaufmann, W., might affect cellular regulators of p53 stability like the adeno- Klingelhutz, A., Seaman, W. T., and Kenney, S. (2002) J. Virol. 76, 12543–12552 virus E1B or human papillomavirus E6 systems. Thus, the 45. Swenson, J. J., Mauser, A. E., Kaufmann, W. K., and Kenney, S. C. (1999) main blocking mechanisms of checkpoint signaling by EBV J. Virol. 73, 6540–6550 would be through regulation of p53. It remains to be clarified 46. Lin, W. C., Lin, F. T., and Nevins, J. R. (2001) Genes Dev. 15, 1833–1844 47. Stevens, C., Smith, L., and La Thangue, N. B. (2003) Nat. Cell Biol. 5, 401–409 whether degradation of p53 is dependent on its binding to the 48. Hammerschmidt, W., and Sugden, B. (1988) Cell 55, 427–433 BZLF1 protein. 49. Tsurumi, T. (2001) Curr. Top. Microbiol. Immunol. 258, 65–87 50. Bell, P., Lieberman, P. M., and Maul, G. G. (2000) J. Virol. 74, 11800–11810 REFERENCES 51. Querido, E., Blanchette, P., Yan, Q., Kamura, T., Morrison, M., Boivin, D., Kaelin, W. G., Conaway, R. C., Conaway, J. W., and Branton, P. E. (2001) 1. Westphal, C. H. (1997) Curr. Biol. 7, R789–R792 2. Shiloh, Y. (2003) Nat. Rev. Cancer 3, 155–168 Genes Dev. 15, 3104–3117

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Mar 4, 2005

There are no references for this article.