Activation of Ataxia Telangiectasia-mutated DNA Damage Checkpoint Signal Transduction Elicited by Herpes Simplex Virus Infection
2005; Elsevier BV; Volume: 280; Issue: 34 Linguagem: Inglês
10.1074/jbc.m500976200
ISSN1083-351X
AutoresNoriko Shirata, Ayumi Kudoh, Tohru Daikoku, Yasutoshi Tatsumi, Masatoshi Fujita, Tohru Kiyono, Yutaka Sugaya, Hiroki Isomura, Kanji Ishizaki, Tatsuya Tsurumi,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoEukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection. Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection. Upon DNA damage, eukaryotic cells exhibit a variety of physiological responses, including cell cycle arrest, activation of DNA repair, and apoptosis. Sets of checkpoint proteins that have been conserved with evolution are rapidly induced to prevent replication or segregation of damaged DNA before repair is completed. Related phosphatidylinositol 3-like kinases, ataxia telangiectasia-mutated (ATM) 1The abbreviations used are: ATM, ataxia telangiectacia-mutated; ATR, ATM-Rad3-related; DSB, DNA double strand breaks; FISH, fluorescence in situ hybridization; HFF, human foreskin fibroblast; HSV, herpes simplex virus; hTERT, human telomerase reverse transcriptase gene; HU, hydroxyurea; IR, ionizing radiation; PBS, phosphate-buffered saline; MRN, complex composed of Mre11, Rad50, and Nbs1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; shRNA, short hairpin RNA; GFP, green fluorescent protein; m.o.i., multiplicity of infection; ACV, acyclovir; PAA, phosphonoacetic acid; BrdUrd, bromodeoxyuridine; p.i., postinfection. and ATM-Rad3-related (ATR), respond to a variety of abnormal DNA structures and initiate signaling cascades leading to a DNA damage checkpoint (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). For example, ATM responds to the presence of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar). On the other hand, the ATR pathway can be stimulated by hydroxyurea (HU), UV light, and base-damaging agents that interfere with the movement of replication forks (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The ATR pathway also responds to DSBs but more slowly than ATM (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). A variety of checkpoint proteins have been identified as substrates for ATM and ATR kinases, including the checkpoint kinases Chk1 and Chk2, as well as p53 (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar, 5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar). ATM exists as an inactive dimer in the nucleus but undergoes autophosphorylation at Ser-1981 in response to DSBs and dissociates into active monomers (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). ATM phosphorylates Chk2 including Thr-68, followed by Chk2 activation (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 7Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (694) Google Scholar, 8Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar). Chk1 is mainly phosphorylated by ATR in response to UV and HU, leading to a 3–5-fold increase in enzyme activity (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Both Mre11 and Nbs1 are also targets of ATM and possibly ATR (9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar, 10Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 11Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar, 12D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar). The MRN complex consisting of Mre11, Rad50, and Nbs1 has been proposed to facilitate ATM activation (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 14Usui T. Ogawa H. Petrini J.H. Mol. Cell. 2001; 7: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 15Williams B.R. Mirzoeva O.K. Morgan W.F. Lin J. Dunnick W. Petrini J.H. Curr. Biol. 2002; 12: 648-653Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and was recently demonstrated to function upstream of ATM activation as a damage sensor, in addition to acting as an effector of ATM signaling (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar). Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are enveloped double-stranded DNA viruses with genomes of 152 and 155 kbp, respectively (17Roizman B. Knipe D.M. Fields B.N. Knipe D.M. Howley P.M. Griffin D.E. Fields Virology. Fourth Ed. Lippincott Williams & Wilkins, Philadelphia, PA2002: 2399-2459Google Scholar). Upon infection immediate-early gene products are expressed and lead to an ordered cascade of viral early and late gene expression. Viral genome is replicated by viral replication machinery, generating highly branched replication intermediates (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The packaging machinery then cleaves concatemeric DNA to monomeric units, which are packaged into preassembled capsids. In HSV-1, DSBs may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection (19Valyi-Nagy T. Olson S.J. Valyi-Nagy K. Montine T.J. Dermody T.S. Virology. 2000; 278: 309-321Crossref PubMed Scopus (73) Google Scholar, 20Milatovic D. Zhang Y. Olson S.J. Montine K.S. Roberts II, L.J. Morrow J.D. Montine T.J. Dermody T.S. Valyi-Nagy T. J. Neurovirol. 2002; 8: 295-305Crossref PubMed Scopus (47) Google Scholar). DSBs are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization (21Huang K.J. Zemelman B.V. Lehman I.R. J. Biol. Chem. 2002; 277: 21071-21079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22Wohlrab F. Chatterjee S. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6432-6436Crossref PubMed Scopus (22) Google Scholar). It is of interest to determine whether host cells can monitor HSV infection as DNA damage. We show here that HSV infection elicits a cellular DNA damage response dependent on ATM. Thereby, DNA damage sensor MRN complex and phosphorylated ATM are recruited to viral replication compartments, presumably recognizing newly synthesized viral DNAs as abnormal DNA structures. Cells—Human foreskin fibroblast (HFF) cells and African green monkey kidney (Vero) cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% fetal calf serum (FCS). Skin fibroblasts from ataxia telangiectasia patients were immortalized by introduction of the human telomerase reverse transcriptase (hTERT) gene (AT1OS/T-n cells) (23Nakamura H. Fukami H. Hayashi Y. Kiyono T. Nakatsugawa S. Hamaguchi M. Ishizaki K. J. Radiat. Res. (Tokyo). 2002; 43: 167-174Crossref PubMed Scopus (30) Google Scholar). Skin fibroblast (NBS1129W) from a NBS patient with a homozygous 5 bp deletion in the NBS gene (gift from Dr. Komatsu, Kyoto University) were similarly immortalized by introduction of hTERT gene as described previously (23Nakamura H. Fukami H. Hayashi Y. Kiyono T. Nakatsugawa S. Hamaguchi M. Ishizaki K. J. Radiat. Res. (Tokyo). 2002; 43: 167-174Crossref PubMed Scopus (30) Google Scholar) to give NBS1129W/T-n. Immortalization of cells with the hTERT gene should not change their original characteristics. AT1OS/T-n and NBS1129W/T-n cells were cultured in DMEM supplemented with 10% FCS and G418 (200 μg/ml), while 293T cells introduced with ATM shRNA (293T-ATM shRNA) or control vector (293T-Control vector) were maintained in DMEM supplemented with 10% FCS and hygromycin B (100 μg/ml). All human cells were used under Japanese ethical guidelines for use of human subjects. Viruses—The HSV-1 strain 17 and the HSV-2 strain 186 were propagated on Vero cells for titration. To inactivate HSV-2 by UV light, a 1-ml volume of HSV stock in a 60-mm dish was exposed to UV from a Toshiba GL-15 bulb at a dose rate of 20 × 106 J/mm2/s for 10 min. The fHSVpac bacterial artificial chromosome and pHGCX plasmid containing the enhanced GFP gene (24Saeki Y. Ichikawa T. Saeki A. Chiocca E.A. Tobler K. Ackermann M. Breakefield X.O. Fraefel C. Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (191) Google Scholar) were kindly provided from Dr. Y. Saeki (Harvard Medical School). A replication-incompetent HSV amplicon expressing GFP was prepared by cotransfection of both bacterial artificial chromosome and plasmid DNAs into Vero2/2 cells and titrated by using 293T cells as described (24Saeki Y. Ichikawa T. Saeki A. Chiocca E.A. Tobler K. Ackermann M. Breakefield X.O. Fraefel C. Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (191) Google Scholar). Infections—Infections were performed on monolayers of cultured cells at indicated multiplicity of infection (m.o.i.). After 1 h at 37 °C, monolayers were overlaid with DMEM containing 10% FCS. Acyclovir (ACV) was used at a final concentration of 100 μg/ml. Phosphonoacetic acid (PAA) was used at a final concentration of 400 μg/ml (25Daikoku T. Kudoh A. Fujita M. Sugaya Y. Isomura H. Shirata N. Tsurumi T. J. Virol. 2005; 79: 3409-3418Crossref PubMed Scopus (74) Google Scholar). The drug was added with the virus and left in for the duration of infection. Antibodies—The anti-UL42 gene product-specific rabbit polyclonal antibody was kindly provided by Dr. Nishiyama, Nagoya University School of Medicine. Anti-Nbs1 and anti-Mre11 antibodies were purchased from BD Biosciences and anti-phospho-ATM (Ser-1981), antiphospho-Chk2 (Thr-68), anti-phospho-Chk1 (Ser-317 and Ser-345), and anti-phospho-p53 (Ser-15) antibodies were from Cell Signaling Technology. Anti-p53 (Ab-6) and anti-ATM (Ab-3) were from Oncogene. Anti-GFP antibody and highly cross-absorbed secondary reagents for dualcolor detection (Alexa 488 and 594) were from Molecular Probes. Immunoblot Analysis—Cells were washed with phosphate-buffered saline (PBS), and treated with lysis buffer (0.02% SDS, 0.5% Triton X-100, 300 mm NaCl, 20 mm Tris-HCl (pH 7.6), 1 mm EDTA, 1 mm dithiothreitol) including a protease inhibitor mixture (Sigma), 200 μm sodium vanadate, and 20 mm sodium fluoride for 20 min on ice. Equal amounts of proteins (30 μg) were separated on SDS-10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Reaction with antibodies and detection with an enhanced chemiluminescence detection system (Amersham Biosciences) were performed as described previously (26Kudoh A. Fujita M. Kiyono T. Kuzushima K. Sugaya Y. Izuta S. Nishiyama Y. Tsurumi T. J. Virol. 2003; 77: 851-861Crossref PubMed Scopus (85) Google Scholar). Immunofluorescence Analysis—Cells grown in culture chambers (Nunc) were washed twice with ice-cold PBS and then treated twice with 0.5% Triton X-100-mCSK buffer (27Daikoku T. Kudoh A. Fujita M. Sugaya Y. Isomura H. Tsurumi T. J. Biol. Chem. 2004; 279: 54817-54825Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) containing a protease inhibitor mixture (Sigma) on ice, followed by fixation with 70% methanol overnight at 4 °C. The fixed cells were blocked for 1 h with 10% FCS in PBS and incubated overnight at 4 °C with individual primary monoclonal antibodies. The cells were incubated with the anti-HSV2 UL42 protein-specific rabbit polyclonal antibody for 15 min at room temperature then incubated for 1 h with secondary goat anti-rabbit or mouse IgG antibodies conjugated with Alexa 488 or 594 (Molecular Probes) and mounted in Vectashield (Vector Laboratories). Image acquisition was performed as described previously (27Daikoku T. Kudoh A. Fujita M. Sugaya Y. Isomura H. Tsurumi T. J. Biol. Chem. 2004; 279: 54817-54825Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The anti-UL42 polyclonal antibody was used at a dilution of 1:5000; all other primary antibodies were employed at 1:100 dilution and the secondary antibodies at 1:250 dilution. Newly synthesized DNAs were labeled by incubating cells with culture medium containing 10 μm BrdUrd for 1 h prior to harvesting. Cells were fixed as described above and then treated for 10 min with 2 n HCl containing 0.5% Triton X-100 to expose incorporated BrdUrd residues before blocking, washing and neutralization with 0.1 m sodium tetraborate (pH 9.0) for 5 min. For BrdUrd staining, an Alexa Fluor 488-conjugated anti-BrdUrd mouse monoclonal antibody (Molecular Probes) was applied. Fluorescence in Situ Hybridization (FISH)—PCR products corresponding to a part of HSV-2 DNA Pol sequence (28Kimura H. Futamura M. Kito H. Ando T. Goto M. Kuzushima K. Shibata M. Morishima T. J. Infect Dis. 1991; 164: 289-293Crossref PubMed Scopus (178) Google Scholar) were labeled with Chroma Tide Alexa Fluor 594-5-dUTP (Molecular Probes) by random primer labeling and used for detection of amplified HSV genomes. At first immunostaining was performed as described above, and then the samples were refixed in 4% paraformaldehyde to cross-link bound antibodies. After permeabilizing in 0.2% Triton X-100 (20 min on ice), cells were digested with RNase A, dehydrated in ethanol, air-dried, and immediately covered with a probe mixture containing 50% formamide in 2× SSC containing probe DNA (10 ng/μl), 10% dextran sulfate, salmon sperm DNA (0.1 μg/μl), and yeast tRNA (1 μg/μl). Probe and cells were simultaneously heated at 94 °C for 4 min then incubated overnight at 37 °C. After hybridization, specimens were washed at 37 °C with 50% formamide in 2× SSC (twice for 15 min each) and 2× SSC. Finally, cells were equilibrated in PBS and mounted in Vectashield (Vector Laboratories). Measurements of Viral Growth Kinetics—293T-ATM shRNA or 293T-Control shRNA cells were infected with HSV-2 186 strain at an m.o.i. of 1 or 0.01 and harvested at the indicated hours postinfection by scraping into the medium and frozen at -80 °C. After thawing, the lysate was sonicated for 1 min on ice and virus yields were titrated by plaque assay on Vero cells (29Nishiyama Y. Rapp F. Virology. 1981; 110: 466-475Crossref PubMed Scopus (35) Google Scholar). Preparation of ATM-silencing 293T Cells—For silencing ATM, the 19-nucleotide sequence corresponding to ATM cDNA nucleotides 604–622 were expressed as shRNA by retrovirus vector. 293T cells were infected with the shRNA retroviruses, and shRNA expressing cells were selected in 200 μg/ml hygromycin B. Details of the vector construction and establishment of ATM-silenced 293T cells will be published elsewhere. HSV Infection Induces a Cellular DNA Damage Response—To investigate whether a cellular DNA damage response was induced upon HSV infection, we examined the phosphorylation status of DNA damage-response proteins in HFF cells (Fig. 1). The ATM kinase responds primarily to DSBs, and this pathway can act during all phases of the cell cycle. It has been recently proposed that ATM is usually present as an inactive multimer and this is activated by autophosphorylation at Ser-1981 after DSBs or changes in the chromatin sturucture (30Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). As shown in Fig. 1, A and B, immunoblotting of cell lysates revealed that levels of the phosphorylated form of ATM at Ser-1981 increased upon HSV-2 and HSV-1 infection, although total levels of ATM remained constant throughout infection. The MRN complex consisting of Mre11, Rad50, and Nbs1 has been suggested to act as a damage sensor (12D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar, 16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar), facilitating ATM activation (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). Activated ATM also phosphorylates Nbs1 (10Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 11Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar). As shown in Fig. 1, A and B, increase in levels of the gel retarded form of Nbs1 was observed, this becoming detectable after 8 or 4 h postinfection (p.i.) with HSV-2 or HSV-1 infection, respectively. The retarded form of Nbs1 was phosphorylated as judged by phosphatase treatment (data not shown). The level of Mre11 appeared constant throughout HSV infection, unlike the adenovirus case (16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, 31Stracker T.H. Carson C.T. Weitzman M.D. Nature. 2002; 418: 348-352Crossref PubMed Scopus (423) Google Scholar). In the presence of DSBs, activated ATM is known to phosphorylate Thr-68 on Chk2 (8Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, 32Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar). Immunoblotting with anti-Chk2 Thr-68-specific antibody also showed phosphorylation of Chk2 at Thr-68 in both HSV-2 and HSV-1 infection (Fig. 1, A and B). Furthermore, phosphorylation of p53 at Ser-15, a widely accepted target of ATM kinase activity, was conspicuous at 8 h p.i. and 4 h p.i. on HSV-2 or HSV-1 infection, respectively, well consistent with the recently reported observation that HSV-1 induces phosphorylation of p53 at Ser-15 dependent on ATM (33Boutell C. Everett R.D. J. Virol. 2004; 78: 8068-8077Crossref PubMed Scopus (35) Google Scholar). In general, phosphorylation of p53 leads to its stabilization and results in increase in its levels (34Vousden K.H. Biochim. Biophys. Acta. 2002; 1602: 47-59Crossref PubMed Scopus (304) Google Scholar, 35Bode A.M. Dong Z. Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1028) Google Scholar). Although HSV-1 infection had no significant effect on total protein levels of p53 throughout (Fig. 1). This is also consistent with the previous report showing that the overall p53 levels are not greatly affected in the HSV-1 infection systems so far examined (36Boutell C. Everett R.D. J. Biol. Chem. 2003; 278: 36596-36602Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). All these data clearly indicate that HSV infection induces cellular DNA damage response. In contrast, this was not evident with UV-inactivated HSV-2 (Fig. 1A). UV-treated virus can adsorb and penetrate into the cells (37Johnson D.C. Ligas M.W. J. Virol. 1988; 62: 4605-4612Crossref PubMed Google Scholar), but expression of the UL42 early viral protein (Fig. 1A) and viral DNA replication (data not shown) were inhibited. Furthermore, when HFF cells were infected with a replication-defective virus (HSV amplicon vector) (24Saeki Y. Ichikawa T. Saeki A. Chiocca E.A. Tobler K. Ackermann M. Breakefield X.O. Fraefel C. Hum. Gene Ther. 1998; 9: 2787-2794Crossref PubMed Scopus (191) Google Scholar) at an m.o.i. of 2, the ATM response was abolished completely, although GFP from the amplicon DNA was expressed (Fig. 2A). In the presence of ACV, the ATM response by the HSV infection was not so inhibited (Fig. 2A). ACV triphosphate is incorporated into viral DNA competing with endogenous dGTP and terminates viral DNA synthesis. The chain termination of viral DNA synthesis might produce immature viral DNAs and consequently elicit the ATM DNA damage response (38Tomicic M.T. Bey E. Wutzler P. Thust R. Kaina B. Mutat. Res. 2002; 505: 1-11Crossref PubMed Scopus (44) Google Scholar). In contrast, PAA, a specific inhibitor of the viral DNA polymerase, appeared to block the ATM DNA damage signaling, especially at low multiplicity of infection (0.1 plaque-forming unit per cell) although the UL42 gene product, viral early protein, was significantly expressed (Fig. 2B). However, at high m.o.i. (1 plaque-forming unit per cell), the DNA damage response was induced to some extent even in the presence of PAA. PAA (400 μg/ml) might not inhibit viral DNA replication completely and immature viral DNA synthesis might be recognized by host damage sensors. Overall, these observations support the idea that viral DNA synthesis triggers activation of the DNA damage response upon HSV infection, but we cannot completely deny the possibility that viral gene expression induces the DNA damage response. It should be noted that not only ATM but also ATR kinases phosphorylate Chk2 kinase at Thr-68 and up-regulate its activity (8Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, 32Ahn J.Y. Schwarz J.K. Piwnica-Worms H. Canman C.E. Cancer Res. 2000; 60: 5934-5936PubMed Google Scholar). Phosphorylation of p53 at Ser-15 is also carried out by ATM/ATR kinases (39Celeste A. Fernandez-Capetillo O. Kruhlak M.J. Pilch D.R. Staudt D.W. Lee A. Bonner R.F. Bonner W.M. Nussenzweig A. Nat. Cell Biol. 2003; 5: 675-679Crossref PubMed Scopus (818) Google Scholar, 40Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (868) Google Scholar). Therefore, HSV infection could activate the ATM, ATR, or both. The ATR kinase responds primarily to DNA replication stress during S phase (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). It can also respond to DSBs if within the S phase but less efficiently than ATM. In contrast to the Chk2 case, phosphorylation of Chk1 at Ser-345 is known to be carried out mainly by ATR kinase, leading to its activation (9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Therefore, we examined Chk1 phosphorylation at Ser-345 in HSV-2- and HSV-1-infected cells (Fig. 1). No significant phosphorylation was observed in either case. Treatment of cells with hydroxyurea, a well studied activator of the replication checkpoint, clearly induced phosphorylation of Ser-345 on Chk1, showing the ATR/Chk1 pathway to be intact in the cells. Thus, we conclude that lytic replication with both types 1 and 2 elicits activation of ATM DNA damage checkpoint signaling rather than the ATR pathway that responds to replication stress. Since both HSV-1 and HSV-2 infection displayed similar cellular DNA damage response, all the following experiments were performed with HSV type2. Mre11 and Nbs1 Proteins Constituting the MRN Complex and Phosphorylated ATM Accumulate in Viral Replication Compartments—HSV DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins cluster and viral DNAs are synthesized. The replicating intermediate viral DNAs have large concatemeric and Y- and X-shaped branch structures (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We therefore examined whether ATM was recruited to the viral replication compartments. The HSV-2-infected cells were first extracted with 0.5% Triton X-100-mCSK buffer containing Triton X-100 to solubilize DNA-unbound forms of viral or cellular proteins and then subjected to immunostaining. As shown in Fig. 3, A and B, the UL42 replication protein was localized in distinct sites in the nuclei of HSV-2-infected cells. HSV infection inhibits cellular replicative DNA synthesis and set forward viral DNA replication (41de Bruyn Kops A. Uprichard S.L. Chen M. Knipe D.M. Virology. 1998; 252: 162-178Crossref PubMed Scopus (57) Google Scholar). The staining sites were completely coincided with the localized foci of newly synthesized viral DNA as judged by BrdUrd incorporation and FISH analyses (Fig. 3, A and B). Thus, since the UL42 protein-localized sites represent loci of viral DNA synthesis, UL42 proteins were thereafter used as markers for viral replication compartments. First, we examined whether DNA damage responsive proteins accumulate in such foci after HSV-2 infection. As shown in Fig. 3C, left panels, in the HSV-2-infected HFF cells, ATM phosphorylated at Ser-1981 was found to be resistant to detergent extraction and became colocalized with viral DNAs in the replication compartments. Next, we assessed the effect of HSV-2 infection on the localization of the Mre11 and Nbs1 (Fig. 3C, middle and right panels). IR resulted in distinct staining of Mre11 and Nbs1 in the nuclei as has been reported (42Kobayashi J. Tauchi H. Sakamoto S. Nakamura A. Morishima K. Matsuura S. Kobayashi T. Tamai K. Tanimoto K. Komatsu K. Curr. Biol. 2002; 12: 1846-1851Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 43Desai-Mehta A. Cerosaletti K.M. Concannon P. Mol. Cell. Biol. 2001; 21: 2184-2191Crossref PubMed Scopus (158) Google Scholar), indicating that the MRN complex is activated and retained in the damaged sites. Upon HSV-2 infection, Mre11 and Nbs1 proteins became resistant to detergent treatment and colocalized predominantly in the viral replication compartments represented by the UL42 staining (Fig. 3C, middle and right panels). Once the pools of endogenous Mre11 and Nbs1 protein were concentrated in this way, the associated fluorescence became resistant to extraction with a mild detergent-containing extraction buffer, indicating that the Mre11 and Nbs1 proteins became not only redistributed to, but also retained within, the close vicinity of newly synthesized viral DNA. The ATM and MRN complex might recognize newly synthesized viral genomic DNA in the replication compartments as abnormal DNA structures and bind to them. Alternatively, it is possible that the complex might function as a player of homologous recombination involved in processing of viral genome rather than as a DNA damage sensor. Wilkinson and Weller (4
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