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

Abstract

When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication. When exposed to genotoxic stress, eukaryotic cells demonstrate a DNA damage response with delay or arrest of cell-cycle progression, providing time for DNA repair. Induction of the Epstein-Barr virus (EBV) lytic program elicited a cellular DNA damage response, with activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of the ATM-Rad3-related (ATR) replication checkpoint pathway, in contrast, was minimal. The DNA damage sensor Mre11-Rad50-Nbs1 (MRN) complex and phosphorylated ATM were recruited and retained in viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also became concentrated in replication compartments and physically interacted with viral BZLF1 protein. Despite the activation of ATM checkpoint signaling, p53-downstream signaling was blocked, with rather high S-phase CDK activity associated with progression of lytic infection. Therefore, although host cells activate ATM checkpoint signaling with response to the lytic viral DNA synthesis, the virus can skillfully evade this host checkpoint security system and actively promote an S-phase-like environment advantageous for viral lytic replication. Eukaryotic cells exhibit a variety of physiological responses, including cell cycle arrest, activation of DNA repair and apoptosis, upon DNA damage. Sets of checkpoint proteins that have been conserved with evolution are rapidly induced to prevent replication or segregation of damaged DNA before repair is completed. The related phosphatidylinositol 3-like kinases, ataxia telangiectasia-mutated (ATM) 1The abbreviations used are: ATM, ataxia telangiectasia-mutated; EBV, Epstein-Barr virus; PBS, phosphate-buffered saline; h.p.i., h post-induction; Gy, Gray; ATR, ATM-Rad3-related; DSB, double-stranded break; FISH, fluorescence in situ hybridization; BrdUrd, bromodeoxyuridine; MRN, Mre11-Rad50-Nbs1; mCSK butter, modified cytoskelton butter; Pipes, 1,4-piperazinediethanesulfonic acid. and ATM-Rad3-related (ATR), respond to a variety of abnormal DNA structures and initiate signaling cascades leading to a DNA damage checkpoint (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). ATM responds to the presence of DNA double-strand breaks (DSBs) induced by ionizing radiation (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2178) Google Scholar). On the other hand, the ATR pathway can be stimulated by hydroxyurea, UV light, and base-damaging agents that interfere with the movement of replication forks (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). The ATR pathway also responds to DSBs, but more slowly than ATM (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2673) Google Scholar). A variety of checkpoint proteins have been identified as substrates for ATM and ATR kinases, including the checkpoint kinases Chk1 and Chk2, as well as γH2AX (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1722) Google Scholar) and p53 (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2178) Google Scholar, 6Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (669) Google Scholar). ATM phosphorylates Chk2 at several sites including Thr-68, followed by Chk2 activation (6Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (669) Google Scholar, 7Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (884) Google Scholar, 8Matsuoka 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 (701) Google Scholar, 9Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (265) Google Scholar). Chk1 is phosphorylated at Ser-345 by ATR in response to UV and hydroxyurea, leading to a 3–5-fold increase in Chk1 activity (6Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (669) Google Scholar, 7Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (884) Google Scholar, 10Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (886) Google Scholar). ATM is activated by intermolecular autophosphorylation on Ser-1981 (11Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2750) Google Scholar). Both Mre11 and Nbs1 are also targets of ATM and possibly ATR (10Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (886) Google Scholar, 12Gatei 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 (412) Google Scholar, 13Wu 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 (376) Google Scholar, 14D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (721) Google Scholar). The MRN complex consisting of Mre11, Rad50, and Nbs1 has been proposed to facilitate ATM activation (15Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (854) Google Scholar, 16Usui T. Ogawa H. Petrini J.H. Mol. Cell. 2001; 7: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 17Williams B.R. Mirzoeva O.K. Morgan W.F. Lin J. Dunnick W. Petrini J.H. Curr. Biol. 2002; 12: 648-653Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and recently demonstrated to function upstream of ATM activation as a damage sensor, in addition to acting as an effector of ATM signaling (15Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (854) Google Scholar, 18Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (424) Google Scholar). ATM/ATR-initiated checkpoint signaling induces p53-dependent and p53-independent responses. The p53-dependent cell cycle checkpoint features p21-mediated inactivation of Cdk2/cyclin E (19el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (8054) Google Scholar, 20Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (729) Google Scholar, 21Dulic V. Kaufmann W.K. Wilson S.J. Tlsty T.D. Lees E. Harper J.W. Elledge S.J. Reed S.I. Cell. 1994; 76: 1013-1023Abstract Full Text PDF PubMed Scopus (1448) Google Scholar), while Chk2 inhibits Cdk2/cyclin E activity by of at of (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2673) Google Scholar), in a p53-independent the is for cell cycle progression and the checkpoint in leading to or cell cycle The Epstein-Barr virus (EBV) is a virus that of targets the cell of viral are and is of virus this infection. the as of that are synthesized in S-phase by the host cell replication the of replication N. J. PubMed Google Scholar). cell a of cells that have a of the lytic The of is but of the is of the BZLF1 The BZLF1 with the other viral J. PubMed Google Scholar) and to an of viral and proteins in viral DNA replication and DNA The lytic of DNA replication is on viral replication an a DNA a a and and to be and J. PubMed Google Scholar). lytic replication in sites in replication compartments in viral replication proteins are S. K. T. PubMed Scopus Google Scholar). have demonstrated that of the lytic program in of replication of cellular DNA as well as replication of viral DNA A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar). The of p53 and CDK the lytic while the of and the of increase as lytic The S-phase-like cellular is to be for the of viral and to as and A. T. Y. H. M. T. Y. T. J. PubMed Scopus Google Scholar). is of to host cells can lytic replication as DNA damage or abnormal DNA the checkpoint signaling to or cell cycle arrest and have cells in BZLF1 is the of a A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar). this for the time that of lytic replication a cellular DNA damage response on DNA damage sensor MRN complex and phosphorylated ATM are recruited to viral replication compartments, recognizing newly synthesized viral DNAs as abnormal DNA structures. the ATM checkpoint signaling was at of Therefore, although lytic replication DNA damage response, the virus can skillfully the host response and actively promote an S-phase-like environment advantageous for viral lytic replication. a with A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar), and cells A. T. Y. H. M. T. Y. T. J. PubMed Scopus Google Scholar), were in with of of and lytic a was to the at a of cells were in with were Cell and Chk2, Mre11, and and E and and to and proteins were as A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar). for and were and cells were at the with with phosphate-buffered and with for on and were to the were at for at and cell were for a of proteins were for high proteins or phosphorylated of or were The proteins were as A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar). of proteins was with an system were with Pipes, and for on followed by with for on The cells were with and for in in with a to phosphorylated Ser-1981 of ATM, to Mre11, and and a to phosphorylated of p53 was at in with a to a to and a to BZLF1 was for h at were for h at and were and were in and by fluorescence fluorescence were and a the were at and the at were with in at The of the and of with were When cells were for with of and fluorescence was and also was with the cells were for with to the before The cells were and with for of was in was with and used for the of was as and in to in on cells were with in and with the in DNA DNA and and cells were at for and at were at with in for and cells were in and in were in of and and The were at for at and was of the and of or with for h at were by and with and The were to followed by were with and with and for on followed by at for at of of were with of in a of for h at to E and B. were with and with The were in of of and were for at with of as were by addition of of and the were by followed by of DNA cells were with of in the presence or of and at the DNAs were a of cells and was and of the of viral cell were as A. 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Google Scholar). of lytic cells of proteins of ATM activity as Chk2 at Thr-68, ATM at and Nbs1 at the of viral replication proteins were at that a is in the checkpoint signaling the lytic while ATM signaling is for lytic replication. in to the of on synthesis, cells were with in the presence or of the DNA was the and were in the of the viral DNA was to more than cell in the of by of checkpoint activation induced by lytic replication viral lytic replication at is that ATM DNA damage signaling induced by the lytic replication is at at the of p53 by the BZLF1 of activity by have on viral lytic replication. that ATM signaling is for lytic replication. has been demonstrated that lytic replication induced the ATM DNA damage response, was the the BZLF1 and activated Chk2 is to Cdk2/cyclin E or A activity by of at of (4Zhou B.B. Elledge S.J. 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