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Originally published In Press as doi:10.1074/jbc.M500976200 on June 17, 2005

J. Biol. Chem., Vol. 280, Issue 34, 30336-30341, August 26, 2005
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Activation of Ataxia Telangiectasia-mutated DNA Damage Checkpoint Signal Transduction Elicited by Herpes Simplex Virus Infection*

Noriko Shirata{ddagger}, Ayumi Kudoh{ddagger}, Tohru Daikoku{ddagger}, Yasutoshi Tatsumi§, Masatoshi Fujita§, Tohru Kiyono§, Yutaka Sugaya{ddagger}, Hiroki Isomura{ddagger}, Kanji Ishizaki¶, and Tatsuya Tsurumi{ddagger}||

From the {ddagger}Divisions of Virology and Central Laboratory & Radiation Biology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan and the §Virology Division, National Cancer Center, Chuo-ku, Tokyo 104-0045, Japan

Received for publication, January 26, 2005 , and in revised form, May 31, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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)1 and ATM-Rad3-related (ATR), respond to a variety of abnormal DNA structures and initiate signaling cascades leading to a DNA damage checkpoint (1). For example, ATM responds to the presence of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) (2). 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 (3). The ATR pathway also responds to DSBs but more slowly than ATM (4).

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 (2, 5). ATM exists as an inactive dimer in the nucleus but undergoes autophosphorylation at Ser-1981 in response to DSBs and dissociates into active monomers (1). ATM phosphorylates Chk2 including Thr-68, followed by Chk2 activation (5–8). Chk1 is mainly phosphorylated by ATR in response to UV and HU, leading to a 3–5-fold increase in enzyme activity (5, 6, 9). Both Mre11 and Nbs1 are also targets of ATM and possibly ATR (9–12). The MRN complex consisting of Mre11, Rad50, and Nbs1 has been proposed to facilitate ATM activation (13–15) and was recently demonstrated to function upstream of ATM activation as a damage sensor, in addition to acting as an effector of ATM signaling (13, 16).

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 (17). 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 (18). 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 (19, 20). DSBs are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization (21, 22).

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.



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  		FIG. 1.
Activation of DNA damage-responsive proteins in HSV-infected cells. HFF cells were infected with HSV type 2 strain 186 at an m.o.i. of 1 (A) or HSV type 1 strain 17 at an m.o.i. of 5 (B) and harvested at the indicated time points postinfection. Thirty µg of proteins from each sample were separated on a 10% SDS-PAGE gels and applied for immunoblot analyses. HFF cells were also infected with UV-inactivated HSV-2 186 (UV HSV-2) at an m.o.i. of 1 and processed similarly (A). Expression profiles of Nbs1, Mre11, ATM phosphorylated at Ser-1981, ATM, Chk2 phosphorylated at Thr-68, Chk1 phosphorylated at Ser345, p53 phosphorylated at Ser-15, p53, and UL42 were examined by immunoblotting. M, mock infected HFF cells. HU, HFF cells treated with 5 mM of hydroxyurea for 12 h. IR, HFF cells exposed to {gamma} radiation with 10 gray and harvested after 1 h.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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) (23). 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 (23) 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 x 106 J/mm2/s for 10 min. The fHSVpac bacterial artificial chromosome and pHGCX plasmid containing the enhanced GFP gene (24) 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 (24).

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 (25). 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 (26).

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 (27) 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 (27). 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 (28) 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 2x 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 2x SSC (twice for 15 min each) and 2x 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 (29).

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (30). 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 (12, 16), facilitating ATM activation (13). Activated ATM also phosphorylates Nbs1 (10, 11). 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 (16, 31).



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  		FIG. 2.
ATM damage responses in cells infected with replication-defective virus or with HSV-2 in the presence of viral DNA replication inhibitors. A, HFF cells were infected with HSV-2 at an m.o.i. of 1 in the presence of ACV or with HSV amplicon vector at an m.o.i. of 2. Cells were harvested at 24 h p.i. B, HFF cells were infected with HSV-2 at an m.o.i. of 1 or 0.1 in the presence (+) or absence (-) of PAA and harvested at 24 h p.i. Thirty µg of proteins from each sample were separated on a 10% SDS-PAGE gels and applied for immunoblot analyses. Expression profiles of Nbs1, ATM phosphorylated at Ser-1981, GFP, and UL42 protein were examined by immunoblotting. Open or Closed arrows indicate phosphorylated or unphosphorylated Nbs1, respectively. Mock, mock infected HFF cells.

 
In the presence of DSBs, activated ATM is known to phosphorylate Thr-68 on Chk2 (8, 32). 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 (33). In general, phosphorylation of p53 leads to its stabilization and results in increase in its levels (34, 35). 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 (36). 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 (37), 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) (24) 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 (38). 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 (8, 32). Phosphorylation of p53 at Ser-15 is also carried out by ATM/ATR kinases (39, 40). Therefore, HSV infection could activate the ATM, ATR, or both. The ATR kinase responds primarily to DNA replication stress during S phase (3). 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 (9). 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 (18). 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 (41). 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 (42, 43), 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 (44) have previously reported that Nbs1 colocalized predominantly with UL29 single-stranded DNA binding protein in HSV-1 replication compartments, well consistent with our observation.



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  		FIG. 3.
ATM and the MRN complex accumulate at sites of viral replication. A and B, architecture of viral replication compartments. HFF cells were infected with HSV-2 186 strain and harvested at 24 h p.i. Newly synthesized DNAs were labeled by incubation with 10 µM BrdUrd added directly to the incubation medium for 1 h prior to harvesting. Cells were treated with 0.5% Triton X-100-mCSK buffer and then fixed with methanol. Viral replication proteins (UL42 protein), newly synthesized DNA (A), and viral DNA (B) were visualized by immunofluorescence, BrdUrd staining, and FISH analyses, respectively, immunostained cells being processed for confocal microscope analyses. The right panels are merged images. C, localization of ATM phosphorylated at Ser-1981, Mre11, and Nbs1. Cells were infected with HSV-2, harvested at 24 h p.i., and extracted with 0.5% Triton X-100-mCSK buffer. Nonionic detergent-extracted cells were then fixed with methanol, double immunostained with the indicated antibodies, and applied for confocal laser microscopy. Shown are profiles of UL42 (red) and ATM phosphorylated at Ser-1981(ATM), Mre11, or Nbs1 (green) proteins. Viral replication compartments were localized by staining for UL42 protein. The right panels are merged images. Mock, mock infected HFF cells. IR, HFF cells exposed to{gamma} radiation with 10 gray and harvested 1 h posttreatment.

 
DNA Damage Responses Induced by HSV Infection Occur in Nbs1-deficient Fibroblasts but Not ATM-deficient Cells—To determine the roles of ATM and Nbs1 in DNA damage responses upon HSV infection, phosphorylation of substrates during infection with the HSV-2 was examined in cells deficient for ATM or Nbs1 (Fig. 4). In ATM-deficient AT1OS/T-n cells, the mobility of Nbs1 was unchanged throughout, implying abrogation of Nbs1 phosphorylation. Also, phosphorylation of Thr-68 on Chk2 was not observed. Phosphorylation of Ser-15 on p53 was not observed and levels of p53 were rather decreased as viral replication proceeded as was observed in normal HFF cells (Fig. 1A). These results further confirmed activation of ATM DNA damage pathway by HSV infection.

On the other hand, in Nbs1-deficient cells, infection with the HSV-2 led to phosphorylation of protein substrates for ATM kinase activity such as ATM Ser-1981, Chk2 Thr-68, and p53 Ser-15, although being delayed (Fig. 4). The data indicate that ATM DNA damage responses were induced by HSV infection even in the absence of a functional MRN complex but that Nbs1 is required for robust activation of ATM DNA damage response. Although the order in which ATM and MRN complex act in the early phase of the DSB response remains unclear, it has been recently reported that functional MRN is required for ATM activation by DSBs and consequently for timely activation of ATM-mediated pathways (13).



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  		FIG. 4.
Expression profiles of DNA damage-responsive proteins in ATM- or Nbs1-defective cell lines infected with HSV. ATM-defective AT1OS/T-n cells or Nbs1-defective NBS1129W/T-n cells were infected with HSV-2 and harvested at the indicated times post infection. Thirty µg of proteins of each sample were separated on a 10% SDS-PAGE gels and applied for immunoblotting. Expression profiles of Nbs1, Mre11, ATM phosphorylated at Ser-1981, ATM, Chk2 phosphorylated at Thr-68, p53 phosphorylated at Ser-15, p53, and UL42 were examined. M, mock infected cells.

 
Formation of Nbs1 Foci at Viral Replication Compartments Is Independent of ATM DNA Damage Signaling Events—We further examined whether accumulation of MRN complexes at viral replication compartments is dependent on the ATM kinase activity. As shown in Fig. 5A, ionic radiation of AT1OS/T-n cells resulted in accumulation of Nbs1 to the damaged nuclei, consistent with previous reports of accumulation of MRN complexes at sites of damage independent of ATM in response to DSBs (39, 45). Similarly, when AT1OS/T-n cells were infected with HSV-2, recruitment of Nbs1 to viral replication compartments occurred (Fig. 5A, right panels). The staining pattern of Nbs1 completely coincided with that for the UL42 viral replication protein, representing the sites of viral DNA synthesis. These findings suggest that recruitment of the MRN complex to viral replication compartments is independent of ATM activation. Furthermore, to determine whether ATM accumulates at replication compartments in the absence of a functional MRN complex, NBS1129W/T-n cells were infected with HSV-2 (Fig. 5B). HSV-2 infection resulted in recruitment of phospho-ATM to the sites of viral replication compartments, demonstrating the ATM kinase to be clearly activated even in the absence of a functional MRN complex.



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  		FIG. 5.
Formation of Nbs1 foci at viral replication compartments is independent of ATM DNA damage signaling events. AT1OS/T-n and NBS1129W/T-n cells were infected with HSV-2 and harvested at 24 h p.i. and extracted with 0.5% Triton X-100-mCSK buffer. The nonionic detergent-extracted cells were then fixed with methanol, double immunostained with indicated antibodies, and applied for confocal laser microscopy. Shown are profiles of UL42 (red) and ATM phosphorylated at Ser-1981(ATM) or Nbs1 (green) proteins. Viral replication compartments were localized by staining for UL42 protein. Mock, mock infected cells. IR, cells exposed to 10 Gy {gamma} radiation and harvested at 1 h posttreatment.

 
ATM-dependent DNA Damage Responses Induced by HSV Infection Do Not Affect Viral Replication—To gain some insights regarding the effect of ATM activation on HSV DNA replication, we introduced expression construct of shRNA targeting ATM gene or control vector into 293T cells using a retrovirus vector and established stably expressing cell lines. It should be noted that in 293T cells p53 activity is suppressed by adenovirus E1B protein, so downstream pathways (such as p21 induction) are not activated. As shown in Fig. 6A, expression of ATM protein, but not ATR (data not shown), was specifically reduced about 90%. Also, ATM silencing caused a considerable reduction in phosphorylation of Nbs1 by HSV-2 infection as judged by mobility on SDS-PAGE. The 293T cells with silenced ATM were then infected with HSV-2 at high or low m.o.i. and analyzed the production of infectious viruses. As shown in Fig. 6B, one-step growth experiments revealed that the growth of HSV-2 was unchanged between ATM-intact and ATM-silenced 293T cells at a high multiplicity of infection. Since we cannot preclude the possibility that defective infectious virus particles might affect viral growth, we also performed HSV infection at low m.o.i. Again the growth of HSV-2 remained unchanged (Fig. 6C). Therefore, we conclude that activated ATM dependent DNA damage signaling is not absolutely required for HSV DNA replication and infectious virus production.



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  		FIG. 6.
ATM activation has no effect on HSV replication. A, 293T cell lines introduced with ATM shRNA (293T-ATM shRNA) or control vector (293T-Control vector) by retrovirus vectors were infected with HSV-2 strain 186 at an m.o.i. of 1 and harvested at the indicated times postinfection. Expression profiles of Nbs1, Mre11, ATM phosphorylated at Ser-1981, and ATM were examined by immunoblotting. M, mock infected cells. HU, cells treated with 5 mM of hydroxyurea for 12 h. IR, cells exposed to {gamma} radiation with 10 Gy and harvested at 1 h posttreatment. B, viral growth in cells infected with HSV-2 strain 186 at high m.o.i. The 293T-ATM shRNA cells (closed circles) and 293T-control vector (open circles) cells were infected with HSV-2 at an input m.o.i. of 1 and harvested at indicated times postinfection with supernatant media. The virus titer was determined by plaque assay on Vero cells. The yields displayed are representative of three separate experiments. C, viral growth in cells infected with HSV-2 strain 186 at an input m.o.i. of 0.01. Titration was carried out as described for B.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We could clearly demonstrate in the present study that the ATM-dependent DNA damage signaling is indeed elicited by HSV infection, probably through replication stress during viral genome synthesis. The MRN complex functions in a common pathway with ATM (11), activating ATM as a damage sensor, in addition to acting as an effector of ATM signaling (16). Clustering of ATM, Mre11, and Nbs1 proteins to HSV replication compartments strongly suggests that these damage sensors recognize newly synthesized viral DNAs as abnormal DNA structures. Infection with an E4-deleted adenovirus results in synthesis of end-joined large viral genomes that are recognized as abnormal DNA structures by the MRN complex, leading to activation of cell cycle checkpoints (31). As a result, ATM damage response signaling is activated, and Chk1, Chk2, 53BP1, p53, Nbs1, and ATM are phosphorylated (16, 31). Similarly, intermediate HSV DNA replication products are thought to be large head-to-tail concatemers with branched structures that might be generated by homologous recombination coupled with replication events (18, 46). When cesium chloride-banded viral DNA is viewed under an electron microscope, a number of unusual structures are evident, including large tangled masses similar to those in replicating DNA from bacteriophage T4, as well as Y-shaped structures and replication bubbles (47). Overall, our present data support the idea that newly synthesized viral DNA is recognized by cellular DNA damage sensors.

It has been suggested that recombination plays a role in the generation of viral replication intermediates (48, 49). In homologous recombination-mediated repair, the MRN complex, most likely with help of other nuclease, might resect the DNA to provide ssDNA overhangs necessary for DNA pairing and strand exchange, the MRN complex only possessing 3'-5' exonuclease activity. This step could be controlled by ATM, since Nbs1 is part of the MRN complex and is a direct substrate for ATM phosphorylation (2, 50). The RAD50 subunit of MRN has ATPase activity that is believed to facilitate DNA unwinding (50, 51). We speculate that the recruited MRN complex might be positively involved in recombination of the viral DNA. This would be studied in the future.

Although ATM/ATR kinase phosphorylates Chk2 at Thr-68 and p53 at Ser-15, the ATR kinase predominantly targets Chk1 at Ser-345 leading to increased Chk1 activity (5, 6, 9). Surprisingly, we could not detect any phosphorylation of Ser-345 on Chk1 in the present study (Fig. 1). The ATR kinase responds primarily to chromosomal DNA replication stress during S phase (3). Our results indicate that HSV infection preferentially activates the ATM DNA damage response without ATR signaling as is the case with reactivation of Epstein-Barr virus lytic replication from latent infection (52). After 24 h p.i., some cells were exhibited BrdUrd staining throughout the nuclei but they did not exhibit any viral replication compartments stained with specific antibodies to the UL42 protein in such cells, suggesting that HSV DNA replication might not occur in S phase cells in which chromosomal DNA replication has already started. If HSV replication actively arrests fork movements of chromosomal DNA replication, ATR DNA damage checkpoint could be activated. HSV replication might occur in G1 or G1/S boundary arrested circumstance, consistent with the previous reports of an association with low CDK2 kinase activity and hypophosphorylated retinoblastoma protein (53).

During wild-type adenovirus infection, the E1b55k/E4orf6 complex degrades the MRN complex, preventing signaling through both ATM and ATR pathways (16, 31). Also, the complex could function in the regulation of p53 levels by degradation of p53 through ubiquitination (54). Thus, adenoviruses appear to have evolved double-check mechanisms to block cell cycle checkpoint signaling pathways. Unlike the case with adenoviruses, lytic replication of HSV does not degrade the MRN complex, since the levels of Mre11 and Nbs1 proved to be almost constant throughout infection (Fig. 1). However, the ICP0 viral immediate-early protein has been shown to interact with and ubiquitinate p53 (36). The ubiquitination of p53 by ICP0 might prevent p53 downstream signaling and reduce the levels of MDM2 ubiquitin ligase and p21 with progression of HSV infection. Thus, the main blocking mechanisms of checkpoint signaling by HSV might be through regulation of p53 to prevent triggering of signals for apoptosis. In addition, HSV has been reported to express additional antiapoptotic gene products encoded by the HSV genome such as ICP27, Us3, and Us5 that act downstream of caspase-3 (55–57). Thus, HSV evades host cellular DNA damage responses and apoptosis by multiple mechanisms. In this regard, no apparent change in viral production in ATM-silenced 293T cells may be expected.


   FOOTNOTES
 
* This work was supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, Culture and Technology of Japan (17659138, 16017322, and 15390153 (to T. T.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed. Tel.:/Fax: 81-52-764-2979; E-mail: ttsurumi{at}aichi-cc.jp.

1 The 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. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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