Induction and Utilization of an ATM Signaling Path
http://www.100md.com
病菌学杂志 2005年第20期
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
ABSTRACT
Progression from G1 to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S- and G2-phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G2M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3- to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM –/– p53–/– than in ATM+/+ p53–/– mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1S957AS966A also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.
INTRODUCTION
A productive infection by polyomavirus is accompanied by activation as well as inactivation of tumor suppressor gene pathways. A critical event is the interaction of the large T antigen with pRb, the product of the retinoblastoma tumor suppressor gene. This interaction leads to an override of the G1 checkpoint and the entry of cells into S, thus allowing viral DNA synthesis to occur (17). A productive infection by polyomavirus also results in the induction of p53 and a DNA damage-like response. Interestingly, this induction also depends on a large T-pRb interaction, based presumably on E2F activation and the assembly of initiation complexes on viral DNA with the formation of theta structures (12). The p53 response, although accompanied by the induction of p21CIP1/WAF1 (p21), does not lead to G1 arrest because of the downstream intervention at the pRb checkpoint by large T. The viral replication pathway leading to p53 induction may impose additional events on the infected cell beyond those of entry into S and raises questions about the possible utilization of a DNA damage response-like pathway by the virus.
Previous studies have shown that cells infected by polyomavirus accumulate in S and G2 phase. This has been investigated with various wild-type and mutant strains of virus in both permissive mouse cells and nonpermissive rat cells (29, 43, 47). Earlier studies have also shown that the adult mouse kidney, which is normally nonpermissive, becomes permissive and allows reactivation of persistent infection by polyomavirus following cellular damage induced by glycerol, cisplatin, or hypoxia (2). These results are consistent with a role for induction of a specific stress or DNA damage response in polyomavirus replication under certain conditions.
Results with simian virus 40 (SV40) likewise show that infection is accompanied by the induction of a G2 checkpoint and indicate a possible role of a DNA damage-like response. The replication of SV40 is enhanced by mitomycin C and inhibited by caffeine (28). In SV40-infected cells, mitomycin C treatment leads to an increase in a 5S form of DNA ligase thought to be involved in DNA repair (36). Lytic infection by SV40 results in G2 arrest and a negative regulation of CDC2 by CHK1, a checkpoint kinase downstream of ATR (ATM-Rad3-related kinase) (30, 38). The SV40 T antigen disrupts nuclear DNA repair foci containing MRE11 and interacts with NBS1, a target of ATM (ataxia telangiectasia mutated) and an essential factor in the S-phase checkpoint that senses DNA damage (14, 51). While ATM may play a role in productive SV40 infection, it apparently has no effect on oncogenesis since tumor growth in SV40 large T transgenic mice is unaffected by the absence of ATM (33). Taken together, the results of these studies raise the possibility that polyomaviruses induce and then utilize a stress response to enhance their own replication.
ATM and ATR are important sensors of different forms of genotoxic stress (39, 44). The checkpoint pathways initiated by ATM and ATR can be p53 dependent or p53 independent. Ser15 in p53 and Ser395 in MDM2 are phosphorylated directly by the ATM/ATR kinases, resulting in the activation of p53 (44) and leading principally to a G1-phase checkpoint (23, 55). ATM/ATR kinases phosphorylate numerous other targets involved in the activation of S- and G2-phase checkpoints and DNA repair, including histone H2AX, CHK1 and -2, BRCA1, NBS1, SMC1, and FANC-D2 (25, 26, 39). However, there is no evidence thus far that the ATM/ATR kinases or their downstream pathways are important for polyomavirus infection. The present study was undertaken to test the hypothesis that polyomavirus not only induces ATM-dependent checkpoints but also derives a growth advantage from them.
MATERIALS AND METHODS
Cells and viruses. Primary baby mouse kidney (BMK) cells were prepared from 15-day-old baby mouse kidneys (50). A31 BALB/3T3 mouse fibroblasts were purchased from ATCC and grown in Dulbecco's minimal essential medium (DMEM) containing 10% calf serum. Primary p53+/+ and p53–/– mouse embryo fibroblasts (MEFs) were prepared from embryos produced by crossing p53+/– mice as described previously (13). ATM+/+ p53–/– (AP29) and ATM–/– p53–/– (AP38) cell lines were generously provided by P. Leder (49). M. Kastan kindly provided the SMC1WT and SMC1S957AS966A MEFs (26). MEFs were cultured in DMEM containing 10% fetal calf serum or 10% newborn calf serum. The polyomavirus laboratory strain RA was the wild type (16). BMK and A31 cells were synchronized by serum starvation for 24 to 72 h in DMEM containing 0.5% or 0.2% calf serum, respectively. The percentage of infected cells was determined as described previously (12). Viruses were titrated on UC1B cells.
Analysis of cell cycle. Cells were harvested by trypsinization and frozen in 250 mM sucrose-40 mM sodium citrate-5% dimethyl sulfoxide. For analysis, nuclei were prepared and stained with propidium iodide as described previously (6, 12). Total DNA was analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the programs Cell Quest and ModFit.
Immunoprecipitation and immunoblotting. Lysates were generally prepared with NP-40 lysis buffer (20 mM Tris, pH 8.0, 135 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 10% glycerol, 1% NP-40, 0.1 mM Na3VO4, 50 mM ?-glycerol phosphate, 10 mM NaF, and protease inhibitors [Roche mini-complete]). Lysates for phospho-H2AX (-H2AX) analysis were prepared with sodium dodecyl sulfate (SDS) lysis buffer (0.5 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) and heated to 95°C. For immunoblotting, 70 to 150 μg of protein was analyzed by SDS-polyacrylamide gel electrophoresis. For immunoprecipitation, 0.5 to 1 mg of protein was incubated with antibody at 4°C overnight, and immune complexes were collected with protein A-Sepharose CL-4B (Amersham Pharmacia) and washed three times with NP-40 lysis buffer and three times with phosphate-buffered saline (PBS). The antibodies for immunoblotting were mouse monoclonal anti-CDC2 (17; Santa Cruz), rabbit polyclonal anti-phospho-CDC2 (Tyr15) (Cell Signaling), rabbit polyclonal anti-p21 (C-19; Santa Cruz), mouse monoclonal mpm2 against mitotic HeLa cell lysate (Upstate Biotechnology), rabbit polyclonal anti--H2AX (Ser139) (Upstate Biotechnology), rabbit polyclonal anti-phospho-p53 (Ser15) (Cell Signaling), rabbit polyclonal anti-p53 (FL-393; Santa Cruz), rabbit polyclonal anti-phospho-ATM (Ser1981) (Rockland), rabbit polyclonal anti-ATM (H-248; Santa Cruz), rabbit polyclonal anti-SMC1 (BL308; Bethyl), and rabbit polyclonal anti-phospho-SMC1 (Ser957) (Bethyl). The antibodies for immunoprecipitation were mouse monoclonal anti-cyclin B1 (GNS1; Santa Cruz), rabbit polyclonal anti-cyclin A (H-432; Santa Cruz), and rabbit polyclonal anti-ATM (H-248; Santa Cruz). Protein concentrations in NP-40 lysates and SDS lysates were determined using the Bio-Rad protein assay and the Bio-Rad DC protein assay, respectively.
Immunofluorescence. Infected cells were fixed with 3.7% neutral buffered formalin and permeabilized with 0.3% Triton X-100 in PBS. After being blocked with 5% normal donkey serum, cells were stained for T antigens with polyclonal anti-T ascites (45) followed by fluorescein isothiocyanate-conjugated anti-rat immunoglobulin G (IgG; Jackson ImmunoResearch). Cells were stained for lamin B with goat polyclonal anti-lamin B (M-20; Santa Cruz) followed by rhodamine-conjugated donkey anti-goat IgG. Cells were counterstained with 4',6'-diamidino-2-phenylindole and were analyzed with a Nikon TE300 fluorescence microscope.
ATM kinase assay. The procedures for preparing cell lysates and assaying ATM kinase activity were described by Ziv et al. (56). Briefly, cells were lysed in DM lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% dodecyl maltoside [Sigma], 5 mM EDTA, 50 mM NaF, 100 μM Na3VO4, and protease inhibitors [Roche mini-complete]). Immunoprecipitation was performed with 1 μg of anti-ATM antibody for 2 h at 4°C, and immune complexes were collected and washed three times with lysis buffer and twice with kinase buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM MnCl2, 10% glycerol, 1 mM dithiothreitol, and 100 μM Na3VO4). Reactions were carried out in kinase buffer containing 20 μM ATP, 10 μCi of [-32P]ATP, and 1 μg of PHAS-1 (Stratagene), with or without caffeine, for 15 min at 37°C. The reaction was stopped with SDS sample buffer, boiled for 5 min, and separated by electrophoresis on a 15% SDS-polyacrylamide gel. Gels were stained and dried for autoradiography or phosphorimaging analysis.
Assay for viral DNA. BMK cells in 3.5-cm dishes were infected with polyomavirus at a multiplicity of infection (MOI) of 0.5 to 1. Medium containing caffeine was added after a 1.5-h adsorption period. Viral DNA was extracted by the Hirt procedure (22). Briefly, monolayers were washed with ice-cold PBS and extracted with 1 ml of buffer containing 20 mM Tris, pH 8.0, 10 mM EDTA, and 0.6% SDS, and extracts were precipitated with 300 μl of 5 M NaCl. Following centrifugation, viral DNA in the supernatant was purified by phenol-chloroform extraction followed by precipitation with isopropyl alcohol. Polyomavirus DNA was analyzed by dot blotting or resolved in a 1% agarose gel and probed for Southern analysis with a 32P-labeled cloned polyomavirus genome prepared by random prime labeling (Roche). ImageJ was used to quantitate the viral DNA.
Assay for virus growth. Virus growth curves were carried out to determine the effects of p53, ATM, and SMC1 on virus replication. Virus yields were determined as previously described (21). Briefly, matched p53+/+ and p53–/– MEFs (passage 2), congenically matched ATM+/+ p53–/– and ATM–/– p53–/– MEFs (passage 9), and SMC1 wild-type and SMC1S957AS966A cells were plated at 2 x 104 cells per well in 24-well plates. Infections were done at an MOI of 0.2 to 0.5 PFU per cell. At 24-h intervals after infection, plates were frozen at –20°C, and virus was harvested. The total virus yield was determined by a plaque assay on UC1B cells. Assays were performed in duplicate in triplicate wells.
Assay for DNA replication. The rates of viral and cellular DNA synthesis were assessed by prelabeling the cells with [14C]thymidine followed by pulse labeling with [3H]thymidine. A31 cells (1.5 x 105) were plated in 6-cm dishes in DMEM containing 10% serum and incubated for 48 h. The medium was replaced with DMEM containing 0.5% serum and 10 nCi/ml of [14C]thymidine, and incubation was continued for 24 h. The medium was removed, and cells were infected at an MOI of 50 and fed with DMEM containing 10% serum, with or without 1 mM caffeine. At the indicated times postinfection, the cells were pulse labeled for 60 min with 2.5 μCi/ml of [3H]thymidine. The labeled cells were washed twice with PBS, and DNAs were extracted by the Hirt procedure (22) with 0.4 ml of 20 mM Tris, pH 8.0, 10 mM EDTA, and 0.6% SDS followed by 0.13 ml of 5 N NaCl. Viral and cellular DNAs were separated by centrifugation at 12,000 rpm for 1 h. The pellet was resuspended in 0.5 ml of 0.01 M EDTA, and 0.3 ml of 20% trichloroacetic acid was added to both fractions. After 10 min, aliquots were collected on GF/A (Whatman) filters and washed three times with 10% trichloroacetic acid and once with ethanol. The filters were air dried and assayed for radioactivity in a liquid scintillation counter. The resulting ratios of 3H to 14C were measures of the rates of DNA synthesis.
RESULTS
Polyomavirus induces S- and G2-phase blocks in productively infected cells. To study the effects of virus infection on cell cycle progression, cells were synchronized by serum starvation prior to infection and serum addition. The results of fluorescence-activated cell sorting (FACS) analysis (Fig. 1A and B) show that following virus infection, BMK and A31 cells enter S phase between 15 and 20 h postinfection and either remain in an extended S phase in the case of A31 cells or enter G2 phase in the case of BMK cells. The percentage of total cells in S phase shown in each panel indicates the effect of virus in extending the period of DNA synthesis in both BMK and A31 cells. Toward the end of the growth cycle, BMK and A31 cells undergo cytopathic effects (CPE) accompanied by the appearance of fragments with less than 2N DNA. Results were similar for experiments conducted with high (10%) and low (0.2% to 0.5%) concentrations of serum (data not shown). To compare cell cycle events in virus-infected cells to those stimulated with high serum levels, uninfected control cells were examined. BMK cells were not driven into S phase by serum (Fig. 1A), whereas serum-stimulated A31 cells completed one mitotic cycle in about 30 h and reentered S phase (Fig. 1B). In both cell types, polyomavirus infection induces a prolonged S phase.
Virus-infected cells do not enter M phase. To determine whether virus-infected cells reached late G2 or early M phase, the phosphorylation state of CDC2 was followed. BMK or A31 cells were G1 arrested by serum starvation and infected or not in low-serum medium. Cyclins B1 and A were immunoprecipitated from BMK and A31 extracts prepared at 44 h and 36 h postinfection, respectively, at a time when CPE first became visible (Fig. 2A). In uninfected serum-starved cells, no CDC2 was seen to coprecipitate with cyclins B1 and A, indicating arrest in G1. In virus-infected BMK cells, CDC2 coprecipitated with cyclin B1 but was phosphorylated on Tyr15, which is indicative of an inactive state. Similar results were observed for A31 cells. Interestingly, in both virus-infected BMK and A31 cells, p21 coprecipitated with cyclin A but not with cyclin B1. This is consistent with the known binding preference of p21 for cyclin A/CDKs over cyclin B1/CDC2 and indicates a further inactivation of cyclin A/CDKs (52). In comparison, A31 cells stimulated with 10% serum showed the active unphosphorylated form of CDC2 in complex with cyclin B1 and an absence of p21 immunoprecipitating with cyclin A. The presence of activated CDC2 in serum-stimulated A31 cells correlates with the full cell cycle proliferation of these cells shown in Fig. 1.
Among the hallmarks of mitosis are the appearance of a number of phosphorylated proteins recognized by the mpm2 monoclonal antibody (11) and the breakdown of the nuclear envelope. To further investigate the effect of polyomavirus infection on the mitotic phase of the cell cycle, the levels of mpm2 proteins and the appearance of nuclear membrane breakdown were examined in virus-infected BMK and A31 cells. The results in Fig. 2B show that mpm2 proteins (around 200 kDa) were only modestly elevated in virus-infected A31 cells compared to serum-stimulated cells. The weak signal may reasonably be ascribed to the 10 to 20% of cells which escaped infection and progressed normally through G2 and M.
During nuclear membrane breakdown, lamin B relocates to the cytoplasm (19). BMK cells were examined by double indirect immunofluorescence using anti-T and anti-lamin B antibodies. At 48 h postinfection, when CPE was evident, lamin B remained associated with the nuclear membrane and did not relocate to the cytoplasm (Fig. 2C). A few mitotic figures were observed in uninfected BMK cultures, most likely corresponding to proliferating fibroblasts present in these primary cultures. These results indicate that productive viral infection does not lead to entry into M phase accompanied by nuclear membrane breakdown. As an additional test for the absence of a requirement for M-phase events in virus growth, infected cells were treated with Colcemid, a known tubulin-disrupting agent. Infection by polyomavirus requires the presence of intact microtubules directly following virus uptake into the cell (20), but Colcemid has no effect if added at later times. Virus yields were unaffected by Colcemid added after 8 h postinfection (data not shown).
ATM is activated in virus-infected cells. The extended S phase and absence of M-phase events in virus-infected cells raise the possibility that viral infection is accompanied by ATM-directed intra-S- or G2-phase checkpoints. To test directly whether ATM is activated in virus-infected cells, in vitro kinase assays were performed. As shown in Fig. 3A, the activity of ATM is elevated in extracts prepared from virus-infected BMK cells compared to the uninfected control. The in vitro kinase activity using PHAS-1 as a substrate was inhibited by caffeine at concentrations of <1 mM, which were previously shown to inhibit ATM (41).
Lytic infection by polyomavirus was previously shown to be accompanied by induction of a p53 response with phosphorylation of p53 on serine 18 (12), indicative of an upstream activation of ATM. To gain further support for ATM activation by virus infection, the effects of caffeine on the levels of -H2AX and phospho-p53 were examined. The results in Fig. 3B (upper panel) show that the addition of 3 mM caffeine to virus-infected BMK cells for 1 hour before harvest at either 24, 30, or 40 h postinfection reduced the level of -H2AX in cells roughly 60%. The levels of phospho-p53 and p21 were strongly reduced when caffeine was added at 24, 31, or 45 h postinfection and left in until harvesting at 48 h (Fig. 3B, lower panel). Finally, immunoblot analysis showed that the level of activated ATM (S1981-P) was significantly elevated in extracts of infected compared to uninfected cells (Fig. 3C). Similar results were seen in infected A31 cells (data not shown).
Caffeine reduces viral DNA synthesis without blocking S-phase entrance. To determine whether a caffeine-sensitive pathway is involved in virus growth, infected BMK cells were treated with caffeine immediately after the virus adsorption period. In cells treated with 3 mM caffeine, the level of viral DNA was reduced roughly sixfold (Fig. 4A). In repeated experiments, the decrease in viral DNA varied from three- to sixfold. The effect of caffeine on viral DNA synthesis was accompanied by a decrease in virus yield of a similar or even greater magnitude (Fig. 4B). To rule out the possibility that caffeine was blocking S-phase entry, thereby reducing the virus yield, the percentage of total cells in S phase was determined by FACS analysis. The data in Fig. 4C show that the treatment of infected cells with caffeine did not block either S-phase entry or the appearance of T antigen-positive cells.
To gain further support for the observation that caffeine lowered the yields of viral DNA without inhibiting cellular DNA synthesis, we attempted to estimate the differential effect of caffeine on the rates of incorporation of [3H]thymidine into viral and cellular DNA in infected A31 cells using the Hirt method of separation (22). As shown in Fig. 4D, incorporation into cellular DNA in the presence of caffeine was enhanced, while incorporation into viral DNA in the same cells was inhibited. Similar results were found by using infected BMK cells (data not shown). Caffeine may enhance incorporation into cellular DNA by blocking an ATM-induced replication stress response; such a response, if uninhibited, would slow progression through S to allow for repair. The inhibitory effect of caffeine on viral DNA synthesis implies the need for a component(s) of the stress response downstream of ATM (see below).
ATM and its downstream target SMC1 enhance the growth rate and yield of virus independently of p53. To explore the basis of the virus's dependence on the ATM pathway, we compared the ability of polyomavirus to replicate in MEFs from ATM-p53 double knockout (49) and SMC1 mutant knockin (26) mice. The role of p53 in virus growth was first examined using mouse embryo fibroblasts from p53+/+ or p53–/– mice. Virus growth was not greatly affected by the presence or absence of p53 (Fig. 5A). In contrast, virus replication was reduced approximately 10-fold in ATM–/– p53–/– MEFs compared to ATM+/+ p53–/– controls (Fig. 5B).
Phosphorylation of the cohesin protein SMC1 at serines 957 and 966 by ATM has recently been shown to be required for activation of the ATM-dependent S-phase checkpoint (26). To determine if SMC1 is needed for the ATM-mediated enhancement of virus growth, SMC1WT and SMC1S957AS966A MEFs were infected, and virus yields were measured. Yields of infectious virus were reduced roughly 10-fold in MEFs expressing the phosphorylation-defective SMC1S957AS966A (Fig. 5C). ATM-dependent activation of the S-phase checkpoint involving phosphorylation of SMC1 on serines 957 and 966 thus plays a positive role in virus replication.
Cells expressing mutant SMC1 show reduced yields of viral DNA and an altered cell cycle response to polyomavirus infection. SMC1S957AS966A cells as well as ATM–/– cells are deficient in producing infectious virus. The level of viral DNA synthesized in polyomavirus-infected SMC1 mutant cells was also decreased (Fig. 6A). SMC1S957AS966A MEFs exhibited a defect in the virus-induced S-phase checkpoint, with a decreased accumulation of cells in S compared to cells expressing the wild-type SMC1 protein and a marked increase in apoptosis (Fig. 6B). Comparable sensitivities to cell death were observed for SMC1S957AS966A cells treated with either ionizing radiation or the alkylating agent methyl methanesulfonate (26). Similar but less striking results indicating an inhibition of viral DNA replication and an abnormal cell cycle response were found using infected ATM–/– cells (data not shown). The greater effect on viral DNA synthesis seen in mutant SMC1 cells than in ATM–/– cells may reflect the fact that ATR and kinases other than ATM can also activate SMC1.
DISCUSSION
The induction of cell cycle checkpoints and activation of the ATM- or ATR-dependent pathway have been reported to accompany infection by a number of different viruses. Adenovirus, herpes simplex virus type 1, Epstein-Barr virus, and retroviruses all induce ATM- or ATR-dependent DNA damage responses (5, 27, 34, 40). Adenovirus eludes the host checkpoint responses that would normally be detrimental to virus growth by degrading the MRN (MRE11, RAD50, NBS1) complex (5). In the case of Epstein-Barr virus, p53 downstream signaling is blocked, and the virus induces an advantageous S-phase environment that promotes viral lytic replication (27). In the case of retroviruses, an ATR-dependent DNA damage response triggers the DNA integration process crucial for retroviral replication (9, 10).
Here we have shown that polyomavirus induces an ATM response and an intra-S-phase checkpoint with an accumulation of cells in S and G2, allowing increased production of progeny virus. No evidence was found for M-phase events. The initiating signal leading to ATM activation and to a DNA damage-like response to polyomavirus infection depends on the activation of E2F by a polyomavirus large T interaction with pRb (12). Among the target genes of E2F are several of the factors needed for formation of the viral initiation complex and for replication itself, including RPA, DNA polymerase /primase, PCNA, and topoisomerase II (4, 46). Other E2F targets include ATM itself, as well as other genes involved in checkpoint signaling and DNA damage repair (3, 4). Moreover, the induction of E2F in response to DNA damage is mediated by ATM-dependent phosphorylation of E2F itself (35). Large T-dependent activation of E2F leads to the formation of viral DNA initiation complexes (theta structures) that may be recognized by the cell as "unlicensed" DNA replication and a DNA damage-like signal leading to ATM activation. Activated ATM may then potentiate the DNA damage response by phosphorylating and stabilizing E2F.
Acting directly or through one or more downstream targets, ATM appears to be responsible for the growth advantage to the virus. The inhibition of ATM by caffeine or the use of ATM-knockout cells results in 10-fold reductions in virus yields compared to untreated or normal cells. The incubation of infected cells with caffeine does not block entry into S but exerts a differential effect on viral versus cellular DNA synthesis, inhibiting the latter while allowing the former to proceed normally. The cohesin protein SMC1, a critical target of ATM in double-strand-break DNA repair (26), appears to be an important factor in the ATM-dependent enhancement of polyomavirus DNA replication. This may reflect a role of cohesin in maintaining proper alignment of viral minichromosomes essential for resolution at the termination of viral DNA replication. Such alignment may facilitate the accurate separation of catenated circular daughter molecules during the topoisomerase II-mediated termination reaction (48). Such an action is consistent with current views on the role of cohesin in sister chromatid pairing in homologous recombination and repair (31, 42). The absence of functional SMC1 has a quantitatively greater effect on infectious virus yields than on the synthesis of viral DNA, consistent with the view that SMC1 is required not for the synthesis of viral DNA per se but for the generation of accurately resolved and packageable viral DNA. The proposed role of cohesin in the topoisomerase II-mediated separation of daughter minichromosomes may not apply in the same way to cellular origins, since these are embedded in linear rather than closed circular DNA molecules.
It should be noted that an independent study using UVC radiation showed that an ATM/ATR-dependent checkpoint targets both cellular and SV40 viral replication for inhibition (37). Differences in the multitargeted ATM and ATR pathways as well as differences imposed by the different host cells in the two studies may explain the apparent discrepancy between the inhibitory effect of DNA damage on SV40 replication and the positive role of the ATM pathway on polyomavirus replication. In our study, we did not introduce any external damage signal apart from events imposed by the virus.
Figure 7 depicts several possible mechanisms by which a polyomavirus-induced ATM response may function to increase virus replication. Large T is multiply phosphorylated by CDC2/CDK2 and other unknown kinases. These phosphorylation events regulate large T's function, specifically in viral DNA replication and not cellular DNA synthesis, via an interaction with pRb (7). The direct phosphorylation by ATM or ATR of large T at Ser267 or Ser271 (S-Q sites that conform to the consensus sequence for ATM) (24) may have a regulatory role. A multiply substituted mutant of large T was found to stimulate rather than inhibit viral DNA replication in a large T-driven ori assay (7), but this has not been investigated with single-site mutants in the context of viral infection.
Checkpoint pathways initiated by the activation of ATM (ATR) can be grouped into three branches, namely, a p53/MDM2 branch and branches that go through CHK1/CHK2 and SMC1 (Fig. 7). ATM activates p53 both by direct phosphorylation on Ser15 and by phosphorylation of MDM2, which inhibits the negative regulatory effect of MDM2 on p53 (44). p53 activation leading to G1 arrest and the induction of apoptosis would be detrimental to virus replication. However, the effects of p53 are effectively bypassed through downstream interactions of the large and middle T antigens. G1 arrest is avoided by interactions of large T with pRb (12, 15), and apoptosis is avoided by middle T acting through phosphatidylinositol 3-kinase and AKT (8). The inhibitory interaction of large T with p150SAL2 that has G1 arrest and proapoptotic functions similar to those of p53 may also help to bypass the effects of a p53 response (32). The downstream pathway of ATM via p53 therefore has no apparent consequence on polyomavirus replication. The other pathways, involving CHK1/CHK2 and SMC1, may benefit polyomavirus replication by imposing blocks in S or G2. The phosphorylation of CHK1 and CHK2 leads to S and G2 checkpoints by inhibiting CDC25 phosphatases. In the CHK1/CHK2 branch, ATM activates CHK2 by phosphorylation on Thr68 (1), and ATR phosphorylates CHK1 on serines 317 and 345 (54). The CHK1/CHK2 branch of ATM signaling blocks CDK activity by a p53-independent mechanism. CHK1 and CHK2 act by phosphorylating CDC25A and -C, the phosphatases that remove the inhibitory phosphates from CDK2 and CDC2 (39). By doing so, CDK2 and CDC2 are prevented from stimulating the S- and G2-phase events needed for cell cycle progression. The SMC1 branch of ATM-dependent checkpoint signaling is carried out in S phase by a group of proteins including SMC1, the MRN complex, and BRCA1, with ATM-mediated phosphorylation of SMC1 on serines 957 and 966 being critical, possibly for DNA repair (25, 26, 53).
The inhibition of polyomavirus DNA replication in SMC1 knockin mutant cells suggests an important role for SMC1 phosphorylation in polyomavirus replication. Moreover, the fact that the inhibition is more striking in SMC1S957AS966A than in ATM–/– cells suggests the possibility of an additional pathway such as the ATR pathway for SMC1 phosphorylation in virus-infected cells. Consistent with this is the finding that low-dose UV radiation induces the phosphorylation of SMC1, presumably by ATR (18). Further investigations focusing directly on SMC1 and its role in viral DNA replication will be needed to confirm and extend the present observations.
ACKNOWLEDGMENTS
We thank Philip Leder (Harvard Medical School, Boston, MA) for providing the ATM+/+ p53–/– and ATM–/– p53–/– mouse embryo fibroblasts and Michael Kastan (St. Jude Children's Research Hospital, Memphis, TN) for providing the SMC1WT and SMC1S957AS966A cells. We also thank Jim DeCaprio and Matthew Weitzman for their advice and John Carroll and Lori Cobb-Kreisman for their technical assistance.
This work was supported by grant RO1 CA90992. T.L.B. is a Virginia and D. K. Ludwig Professor of Cancer Research and Teaching.
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ABSTRACT
Progression from G1 to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S- and G2-phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G2M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3- to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM –/– p53–/– than in ATM+/+ p53–/– mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1S957AS966A also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.
INTRODUCTION
A productive infection by polyomavirus is accompanied by activation as well as inactivation of tumor suppressor gene pathways. A critical event is the interaction of the large T antigen with pRb, the product of the retinoblastoma tumor suppressor gene. This interaction leads to an override of the G1 checkpoint and the entry of cells into S, thus allowing viral DNA synthesis to occur (17). A productive infection by polyomavirus also results in the induction of p53 and a DNA damage-like response. Interestingly, this induction also depends on a large T-pRb interaction, based presumably on E2F activation and the assembly of initiation complexes on viral DNA with the formation of theta structures (12). The p53 response, although accompanied by the induction of p21CIP1/WAF1 (p21), does not lead to G1 arrest because of the downstream intervention at the pRb checkpoint by large T. The viral replication pathway leading to p53 induction may impose additional events on the infected cell beyond those of entry into S and raises questions about the possible utilization of a DNA damage response-like pathway by the virus.
Previous studies have shown that cells infected by polyomavirus accumulate in S and G2 phase. This has been investigated with various wild-type and mutant strains of virus in both permissive mouse cells and nonpermissive rat cells (29, 43, 47). Earlier studies have also shown that the adult mouse kidney, which is normally nonpermissive, becomes permissive and allows reactivation of persistent infection by polyomavirus following cellular damage induced by glycerol, cisplatin, or hypoxia (2). These results are consistent with a role for induction of a specific stress or DNA damage response in polyomavirus replication under certain conditions.
Results with simian virus 40 (SV40) likewise show that infection is accompanied by the induction of a G2 checkpoint and indicate a possible role of a DNA damage-like response. The replication of SV40 is enhanced by mitomycin C and inhibited by caffeine (28). In SV40-infected cells, mitomycin C treatment leads to an increase in a 5S form of DNA ligase thought to be involved in DNA repair (36). Lytic infection by SV40 results in G2 arrest and a negative regulation of CDC2 by CHK1, a checkpoint kinase downstream of ATR (ATM-Rad3-related kinase) (30, 38). The SV40 T antigen disrupts nuclear DNA repair foci containing MRE11 and interacts with NBS1, a target of ATM (ataxia telangiectasia mutated) and an essential factor in the S-phase checkpoint that senses DNA damage (14, 51). While ATM may play a role in productive SV40 infection, it apparently has no effect on oncogenesis since tumor growth in SV40 large T transgenic mice is unaffected by the absence of ATM (33). Taken together, the results of these studies raise the possibility that polyomaviruses induce and then utilize a stress response to enhance their own replication.
ATM and ATR are important sensors of different forms of genotoxic stress (39, 44). The checkpoint pathways initiated by ATM and ATR can be p53 dependent or p53 independent. Ser15 in p53 and Ser395 in MDM2 are phosphorylated directly by the ATM/ATR kinases, resulting in the activation of p53 (44) and leading principally to a G1-phase checkpoint (23, 55). ATM/ATR kinases phosphorylate numerous other targets involved in the activation of S- and G2-phase checkpoints and DNA repair, including histone H2AX, CHK1 and -2, BRCA1, NBS1, SMC1, and FANC-D2 (25, 26, 39). However, there is no evidence thus far that the ATM/ATR kinases or their downstream pathways are important for polyomavirus infection. The present study was undertaken to test the hypothesis that polyomavirus not only induces ATM-dependent checkpoints but also derives a growth advantage from them.
MATERIALS AND METHODS
Cells and viruses. Primary baby mouse kidney (BMK) cells were prepared from 15-day-old baby mouse kidneys (50). A31 BALB/3T3 mouse fibroblasts were purchased from ATCC and grown in Dulbecco's minimal essential medium (DMEM) containing 10% calf serum. Primary p53+/+ and p53–/– mouse embryo fibroblasts (MEFs) were prepared from embryos produced by crossing p53+/– mice as described previously (13). ATM+/+ p53–/– (AP29) and ATM–/– p53–/– (AP38) cell lines were generously provided by P. Leder (49). M. Kastan kindly provided the SMC1WT and SMC1S957AS966A MEFs (26). MEFs were cultured in DMEM containing 10% fetal calf serum or 10% newborn calf serum. The polyomavirus laboratory strain RA was the wild type (16). BMK and A31 cells were synchronized by serum starvation for 24 to 72 h in DMEM containing 0.5% or 0.2% calf serum, respectively. The percentage of infected cells was determined as described previously (12). Viruses were titrated on UC1B cells.
Analysis of cell cycle. Cells were harvested by trypsinization and frozen in 250 mM sucrose-40 mM sodium citrate-5% dimethyl sulfoxide. For analysis, nuclei were prepared and stained with propidium iodide as described previously (6, 12). Total DNA was analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the programs Cell Quest and ModFit.
Immunoprecipitation and immunoblotting. Lysates were generally prepared with NP-40 lysis buffer (20 mM Tris, pH 8.0, 135 mM NaCl, 1 mM MgCl2, 0.1 mM CaCl2, 10% glycerol, 1% NP-40, 0.1 mM Na3VO4, 50 mM ?-glycerol phosphate, 10 mM NaF, and protease inhibitors [Roche mini-complete]). Lysates for phospho-H2AX (-H2AX) analysis were prepared with sodium dodecyl sulfate (SDS) lysis buffer (0.5 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) and heated to 95°C. For immunoblotting, 70 to 150 μg of protein was analyzed by SDS-polyacrylamide gel electrophoresis. For immunoprecipitation, 0.5 to 1 mg of protein was incubated with antibody at 4°C overnight, and immune complexes were collected with protein A-Sepharose CL-4B (Amersham Pharmacia) and washed three times with NP-40 lysis buffer and three times with phosphate-buffered saline (PBS). The antibodies for immunoblotting were mouse monoclonal anti-CDC2 (17; Santa Cruz), rabbit polyclonal anti-phospho-CDC2 (Tyr15) (Cell Signaling), rabbit polyclonal anti-p21 (C-19; Santa Cruz), mouse monoclonal mpm2 against mitotic HeLa cell lysate (Upstate Biotechnology), rabbit polyclonal anti--H2AX (Ser139) (Upstate Biotechnology), rabbit polyclonal anti-phospho-p53 (Ser15) (Cell Signaling), rabbit polyclonal anti-p53 (FL-393; Santa Cruz), rabbit polyclonal anti-phospho-ATM (Ser1981) (Rockland), rabbit polyclonal anti-ATM (H-248; Santa Cruz), rabbit polyclonal anti-SMC1 (BL308; Bethyl), and rabbit polyclonal anti-phospho-SMC1 (Ser957) (Bethyl). The antibodies for immunoprecipitation were mouse monoclonal anti-cyclin B1 (GNS1; Santa Cruz), rabbit polyclonal anti-cyclin A (H-432; Santa Cruz), and rabbit polyclonal anti-ATM (H-248; Santa Cruz). Protein concentrations in NP-40 lysates and SDS lysates were determined using the Bio-Rad protein assay and the Bio-Rad DC protein assay, respectively.
Immunofluorescence. Infected cells were fixed with 3.7% neutral buffered formalin and permeabilized with 0.3% Triton X-100 in PBS. After being blocked with 5% normal donkey serum, cells were stained for T antigens with polyclonal anti-T ascites (45) followed by fluorescein isothiocyanate-conjugated anti-rat immunoglobulin G (IgG; Jackson ImmunoResearch). Cells were stained for lamin B with goat polyclonal anti-lamin B (M-20; Santa Cruz) followed by rhodamine-conjugated donkey anti-goat IgG. Cells were counterstained with 4',6'-diamidino-2-phenylindole and were analyzed with a Nikon TE300 fluorescence microscope.
ATM kinase assay. The procedures for preparing cell lysates and assaying ATM kinase activity were described by Ziv et al. (56). Briefly, cells were lysed in DM lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% dodecyl maltoside [Sigma], 5 mM EDTA, 50 mM NaF, 100 μM Na3VO4, and protease inhibitors [Roche mini-complete]). Immunoprecipitation was performed with 1 μg of anti-ATM antibody for 2 h at 4°C, and immune complexes were collected and washed three times with lysis buffer and twice with kinase buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM MnCl2, 10% glycerol, 1 mM dithiothreitol, and 100 μM Na3VO4). Reactions were carried out in kinase buffer containing 20 μM ATP, 10 μCi of [-32P]ATP, and 1 μg of PHAS-1 (Stratagene), with or without caffeine, for 15 min at 37°C. The reaction was stopped with SDS sample buffer, boiled for 5 min, and separated by electrophoresis on a 15% SDS-polyacrylamide gel. Gels were stained and dried for autoradiography or phosphorimaging analysis.
Assay for viral DNA. BMK cells in 3.5-cm dishes were infected with polyomavirus at a multiplicity of infection (MOI) of 0.5 to 1. Medium containing caffeine was added after a 1.5-h adsorption period. Viral DNA was extracted by the Hirt procedure (22). Briefly, monolayers were washed with ice-cold PBS and extracted with 1 ml of buffer containing 20 mM Tris, pH 8.0, 10 mM EDTA, and 0.6% SDS, and extracts were precipitated with 300 μl of 5 M NaCl. Following centrifugation, viral DNA in the supernatant was purified by phenol-chloroform extraction followed by precipitation with isopropyl alcohol. Polyomavirus DNA was analyzed by dot blotting or resolved in a 1% agarose gel and probed for Southern analysis with a 32P-labeled cloned polyomavirus genome prepared by random prime labeling (Roche). ImageJ was used to quantitate the viral DNA.
Assay for virus growth. Virus growth curves were carried out to determine the effects of p53, ATM, and SMC1 on virus replication. Virus yields were determined as previously described (21). Briefly, matched p53+/+ and p53–/– MEFs (passage 2), congenically matched ATM+/+ p53–/– and ATM–/– p53–/– MEFs (passage 9), and SMC1 wild-type and SMC1S957AS966A cells were plated at 2 x 104 cells per well in 24-well plates. Infections were done at an MOI of 0.2 to 0.5 PFU per cell. At 24-h intervals after infection, plates were frozen at –20°C, and virus was harvested. The total virus yield was determined by a plaque assay on UC1B cells. Assays were performed in duplicate in triplicate wells.
Assay for DNA replication. The rates of viral and cellular DNA synthesis were assessed by prelabeling the cells with [14C]thymidine followed by pulse labeling with [3H]thymidine. A31 cells (1.5 x 105) were plated in 6-cm dishes in DMEM containing 10% serum and incubated for 48 h. The medium was replaced with DMEM containing 0.5% serum and 10 nCi/ml of [14C]thymidine, and incubation was continued for 24 h. The medium was removed, and cells were infected at an MOI of 50 and fed with DMEM containing 10% serum, with or without 1 mM caffeine. At the indicated times postinfection, the cells were pulse labeled for 60 min with 2.5 μCi/ml of [3H]thymidine. The labeled cells were washed twice with PBS, and DNAs were extracted by the Hirt procedure (22) with 0.4 ml of 20 mM Tris, pH 8.0, 10 mM EDTA, and 0.6% SDS followed by 0.13 ml of 5 N NaCl. Viral and cellular DNAs were separated by centrifugation at 12,000 rpm for 1 h. The pellet was resuspended in 0.5 ml of 0.01 M EDTA, and 0.3 ml of 20% trichloroacetic acid was added to both fractions. After 10 min, aliquots were collected on GF/A (Whatman) filters and washed three times with 10% trichloroacetic acid and once with ethanol. The filters were air dried and assayed for radioactivity in a liquid scintillation counter. The resulting ratios of 3H to 14C were measures of the rates of DNA synthesis.
RESULTS
Polyomavirus induces S- and G2-phase blocks in productively infected cells. To study the effects of virus infection on cell cycle progression, cells were synchronized by serum starvation prior to infection and serum addition. The results of fluorescence-activated cell sorting (FACS) analysis (Fig. 1A and B) show that following virus infection, BMK and A31 cells enter S phase between 15 and 20 h postinfection and either remain in an extended S phase in the case of A31 cells or enter G2 phase in the case of BMK cells. The percentage of total cells in S phase shown in each panel indicates the effect of virus in extending the period of DNA synthesis in both BMK and A31 cells. Toward the end of the growth cycle, BMK and A31 cells undergo cytopathic effects (CPE) accompanied by the appearance of fragments with less than 2N DNA. Results were similar for experiments conducted with high (10%) and low (0.2% to 0.5%) concentrations of serum (data not shown). To compare cell cycle events in virus-infected cells to those stimulated with high serum levels, uninfected control cells were examined. BMK cells were not driven into S phase by serum (Fig. 1A), whereas serum-stimulated A31 cells completed one mitotic cycle in about 30 h and reentered S phase (Fig. 1B). In both cell types, polyomavirus infection induces a prolonged S phase.
Virus-infected cells do not enter M phase. To determine whether virus-infected cells reached late G2 or early M phase, the phosphorylation state of CDC2 was followed. BMK or A31 cells were G1 arrested by serum starvation and infected or not in low-serum medium. Cyclins B1 and A were immunoprecipitated from BMK and A31 extracts prepared at 44 h and 36 h postinfection, respectively, at a time when CPE first became visible (Fig. 2A). In uninfected serum-starved cells, no CDC2 was seen to coprecipitate with cyclins B1 and A, indicating arrest in G1. In virus-infected BMK cells, CDC2 coprecipitated with cyclin B1 but was phosphorylated on Tyr15, which is indicative of an inactive state. Similar results were observed for A31 cells. Interestingly, in both virus-infected BMK and A31 cells, p21 coprecipitated with cyclin A but not with cyclin B1. This is consistent with the known binding preference of p21 for cyclin A/CDKs over cyclin B1/CDC2 and indicates a further inactivation of cyclin A/CDKs (52). In comparison, A31 cells stimulated with 10% serum showed the active unphosphorylated form of CDC2 in complex with cyclin B1 and an absence of p21 immunoprecipitating with cyclin A. The presence of activated CDC2 in serum-stimulated A31 cells correlates with the full cell cycle proliferation of these cells shown in Fig. 1.
Among the hallmarks of mitosis are the appearance of a number of phosphorylated proteins recognized by the mpm2 monoclonal antibody (11) and the breakdown of the nuclear envelope. To further investigate the effect of polyomavirus infection on the mitotic phase of the cell cycle, the levels of mpm2 proteins and the appearance of nuclear membrane breakdown were examined in virus-infected BMK and A31 cells. The results in Fig. 2B show that mpm2 proteins (around 200 kDa) were only modestly elevated in virus-infected A31 cells compared to serum-stimulated cells. The weak signal may reasonably be ascribed to the 10 to 20% of cells which escaped infection and progressed normally through G2 and M.
During nuclear membrane breakdown, lamin B relocates to the cytoplasm (19). BMK cells were examined by double indirect immunofluorescence using anti-T and anti-lamin B antibodies. At 48 h postinfection, when CPE was evident, lamin B remained associated with the nuclear membrane and did not relocate to the cytoplasm (Fig. 2C). A few mitotic figures were observed in uninfected BMK cultures, most likely corresponding to proliferating fibroblasts present in these primary cultures. These results indicate that productive viral infection does not lead to entry into M phase accompanied by nuclear membrane breakdown. As an additional test for the absence of a requirement for M-phase events in virus growth, infected cells were treated with Colcemid, a known tubulin-disrupting agent. Infection by polyomavirus requires the presence of intact microtubules directly following virus uptake into the cell (20), but Colcemid has no effect if added at later times. Virus yields were unaffected by Colcemid added after 8 h postinfection (data not shown).
ATM is activated in virus-infected cells. The extended S phase and absence of M-phase events in virus-infected cells raise the possibility that viral infection is accompanied by ATM-directed intra-S- or G2-phase checkpoints. To test directly whether ATM is activated in virus-infected cells, in vitro kinase assays were performed. As shown in Fig. 3A, the activity of ATM is elevated in extracts prepared from virus-infected BMK cells compared to the uninfected control. The in vitro kinase activity using PHAS-1 as a substrate was inhibited by caffeine at concentrations of <1 mM, which were previously shown to inhibit ATM (41).
Lytic infection by polyomavirus was previously shown to be accompanied by induction of a p53 response with phosphorylation of p53 on serine 18 (12), indicative of an upstream activation of ATM. To gain further support for ATM activation by virus infection, the effects of caffeine on the levels of -H2AX and phospho-p53 were examined. The results in Fig. 3B (upper panel) show that the addition of 3 mM caffeine to virus-infected BMK cells for 1 hour before harvest at either 24, 30, or 40 h postinfection reduced the level of -H2AX in cells roughly 60%. The levels of phospho-p53 and p21 were strongly reduced when caffeine was added at 24, 31, or 45 h postinfection and left in until harvesting at 48 h (Fig. 3B, lower panel). Finally, immunoblot analysis showed that the level of activated ATM (S1981-P) was significantly elevated in extracts of infected compared to uninfected cells (Fig. 3C). Similar results were seen in infected A31 cells (data not shown).
Caffeine reduces viral DNA synthesis without blocking S-phase entrance. To determine whether a caffeine-sensitive pathway is involved in virus growth, infected BMK cells were treated with caffeine immediately after the virus adsorption period. In cells treated with 3 mM caffeine, the level of viral DNA was reduced roughly sixfold (Fig. 4A). In repeated experiments, the decrease in viral DNA varied from three- to sixfold. The effect of caffeine on viral DNA synthesis was accompanied by a decrease in virus yield of a similar or even greater magnitude (Fig. 4B). To rule out the possibility that caffeine was blocking S-phase entry, thereby reducing the virus yield, the percentage of total cells in S phase was determined by FACS analysis. The data in Fig. 4C show that the treatment of infected cells with caffeine did not block either S-phase entry or the appearance of T antigen-positive cells.
To gain further support for the observation that caffeine lowered the yields of viral DNA without inhibiting cellular DNA synthesis, we attempted to estimate the differential effect of caffeine on the rates of incorporation of [3H]thymidine into viral and cellular DNA in infected A31 cells using the Hirt method of separation (22). As shown in Fig. 4D, incorporation into cellular DNA in the presence of caffeine was enhanced, while incorporation into viral DNA in the same cells was inhibited. Similar results were found by using infected BMK cells (data not shown). Caffeine may enhance incorporation into cellular DNA by blocking an ATM-induced replication stress response; such a response, if uninhibited, would slow progression through S to allow for repair. The inhibitory effect of caffeine on viral DNA synthesis implies the need for a component(s) of the stress response downstream of ATM (see below).
ATM and its downstream target SMC1 enhance the growth rate and yield of virus independently of p53. To explore the basis of the virus's dependence on the ATM pathway, we compared the ability of polyomavirus to replicate in MEFs from ATM-p53 double knockout (49) and SMC1 mutant knockin (26) mice. The role of p53 in virus growth was first examined using mouse embryo fibroblasts from p53+/+ or p53–/– mice. Virus growth was not greatly affected by the presence or absence of p53 (Fig. 5A). In contrast, virus replication was reduced approximately 10-fold in ATM–/– p53–/– MEFs compared to ATM+/+ p53–/– controls (Fig. 5B).
Phosphorylation of the cohesin protein SMC1 at serines 957 and 966 by ATM has recently been shown to be required for activation of the ATM-dependent S-phase checkpoint (26). To determine if SMC1 is needed for the ATM-mediated enhancement of virus growth, SMC1WT and SMC1S957AS966A MEFs were infected, and virus yields were measured. Yields of infectious virus were reduced roughly 10-fold in MEFs expressing the phosphorylation-defective SMC1S957AS966A (Fig. 5C). ATM-dependent activation of the S-phase checkpoint involving phosphorylation of SMC1 on serines 957 and 966 thus plays a positive role in virus replication.
Cells expressing mutant SMC1 show reduced yields of viral DNA and an altered cell cycle response to polyomavirus infection. SMC1S957AS966A cells as well as ATM–/– cells are deficient in producing infectious virus. The level of viral DNA synthesized in polyomavirus-infected SMC1 mutant cells was also decreased (Fig. 6A). SMC1S957AS966A MEFs exhibited a defect in the virus-induced S-phase checkpoint, with a decreased accumulation of cells in S compared to cells expressing the wild-type SMC1 protein and a marked increase in apoptosis (Fig. 6B). Comparable sensitivities to cell death were observed for SMC1S957AS966A cells treated with either ionizing radiation or the alkylating agent methyl methanesulfonate (26). Similar but less striking results indicating an inhibition of viral DNA replication and an abnormal cell cycle response were found using infected ATM–/– cells (data not shown). The greater effect on viral DNA synthesis seen in mutant SMC1 cells than in ATM–/– cells may reflect the fact that ATR and kinases other than ATM can also activate SMC1.
DISCUSSION
The induction of cell cycle checkpoints and activation of the ATM- or ATR-dependent pathway have been reported to accompany infection by a number of different viruses. Adenovirus, herpes simplex virus type 1, Epstein-Barr virus, and retroviruses all induce ATM- or ATR-dependent DNA damage responses (5, 27, 34, 40). Adenovirus eludes the host checkpoint responses that would normally be detrimental to virus growth by degrading the MRN (MRE11, RAD50, NBS1) complex (5). In the case of Epstein-Barr virus, p53 downstream signaling is blocked, and the virus induces an advantageous S-phase environment that promotes viral lytic replication (27). In the case of retroviruses, an ATR-dependent DNA damage response triggers the DNA integration process crucial for retroviral replication (9, 10).
Here we have shown that polyomavirus induces an ATM response and an intra-S-phase checkpoint with an accumulation of cells in S and G2, allowing increased production of progeny virus. No evidence was found for M-phase events. The initiating signal leading to ATM activation and to a DNA damage-like response to polyomavirus infection depends on the activation of E2F by a polyomavirus large T interaction with pRb (12). Among the target genes of E2F are several of the factors needed for formation of the viral initiation complex and for replication itself, including RPA, DNA polymerase /primase, PCNA, and topoisomerase II (4, 46). Other E2F targets include ATM itself, as well as other genes involved in checkpoint signaling and DNA damage repair (3, 4). Moreover, the induction of E2F in response to DNA damage is mediated by ATM-dependent phosphorylation of E2F itself (35). Large T-dependent activation of E2F leads to the formation of viral DNA initiation complexes (theta structures) that may be recognized by the cell as "unlicensed" DNA replication and a DNA damage-like signal leading to ATM activation. Activated ATM may then potentiate the DNA damage response by phosphorylating and stabilizing E2F.
Acting directly or through one or more downstream targets, ATM appears to be responsible for the growth advantage to the virus. The inhibition of ATM by caffeine or the use of ATM-knockout cells results in 10-fold reductions in virus yields compared to untreated or normal cells. The incubation of infected cells with caffeine does not block entry into S but exerts a differential effect on viral versus cellular DNA synthesis, inhibiting the latter while allowing the former to proceed normally. The cohesin protein SMC1, a critical target of ATM in double-strand-break DNA repair (26), appears to be an important factor in the ATM-dependent enhancement of polyomavirus DNA replication. This may reflect a role of cohesin in maintaining proper alignment of viral minichromosomes essential for resolution at the termination of viral DNA replication. Such alignment may facilitate the accurate separation of catenated circular daughter molecules during the topoisomerase II-mediated termination reaction (48). Such an action is consistent with current views on the role of cohesin in sister chromatid pairing in homologous recombination and repair (31, 42). The absence of functional SMC1 has a quantitatively greater effect on infectious virus yields than on the synthesis of viral DNA, consistent with the view that SMC1 is required not for the synthesis of viral DNA per se but for the generation of accurately resolved and packageable viral DNA. The proposed role of cohesin in the topoisomerase II-mediated separation of daughter minichromosomes may not apply in the same way to cellular origins, since these are embedded in linear rather than closed circular DNA molecules.
It should be noted that an independent study using UVC radiation showed that an ATM/ATR-dependent checkpoint targets both cellular and SV40 viral replication for inhibition (37). Differences in the multitargeted ATM and ATR pathways as well as differences imposed by the different host cells in the two studies may explain the apparent discrepancy between the inhibitory effect of DNA damage on SV40 replication and the positive role of the ATM pathway on polyomavirus replication. In our study, we did not introduce any external damage signal apart from events imposed by the virus.
Figure 7 depicts several possible mechanisms by which a polyomavirus-induced ATM response may function to increase virus replication. Large T is multiply phosphorylated by CDC2/CDK2 and other unknown kinases. These phosphorylation events regulate large T's function, specifically in viral DNA replication and not cellular DNA synthesis, via an interaction with pRb (7). The direct phosphorylation by ATM or ATR of large T at Ser267 or Ser271 (S-Q sites that conform to the consensus sequence for ATM) (24) may have a regulatory role. A multiply substituted mutant of large T was found to stimulate rather than inhibit viral DNA replication in a large T-driven ori assay (7), but this has not been investigated with single-site mutants in the context of viral infection.
Checkpoint pathways initiated by the activation of ATM (ATR) can be grouped into three branches, namely, a p53/MDM2 branch and branches that go through CHK1/CHK2 and SMC1 (Fig. 7). ATM activates p53 both by direct phosphorylation on Ser15 and by phosphorylation of MDM2, which inhibits the negative regulatory effect of MDM2 on p53 (44). p53 activation leading to G1 arrest and the induction of apoptosis would be detrimental to virus replication. However, the effects of p53 are effectively bypassed through downstream interactions of the large and middle T antigens. G1 arrest is avoided by interactions of large T with pRb (12, 15), and apoptosis is avoided by middle T acting through phosphatidylinositol 3-kinase and AKT (8). The inhibitory interaction of large T with p150SAL2 that has G1 arrest and proapoptotic functions similar to those of p53 may also help to bypass the effects of a p53 response (32). The downstream pathway of ATM via p53 therefore has no apparent consequence on polyomavirus replication. The other pathways, involving CHK1/CHK2 and SMC1, may benefit polyomavirus replication by imposing blocks in S or G2. The phosphorylation of CHK1 and CHK2 leads to S and G2 checkpoints by inhibiting CDC25 phosphatases. In the CHK1/CHK2 branch, ATM activates CHK2 by phosphorylation on Thr68 (1), and ATR phosphorylates CHK1 on serines 317 and 345 (54). The CHK1/CHK2 branch of ATM signaling blocks CDK activity by a p53-independent mechanism. CHK1 and CHK2 act by phosphorylating CDC25A and -C, the phosphatases that remove the inhibitory phosphates from CDK2 and CDC2 (39). By doing so, CDK2 and CDC2 are prevented from stimulating the S- and G2-phase events needed for cell cycle progression. The SMC1 branch of ATM-dependent checkpoint signaling is carried out in S phase by a group of proteins including SMC1, the MRN complex, and BRCA1, with ATM-mediated phosphorylation of SMC1 on serines 957 and 966 being critical, possibly for DNA repair (25, 26, 53).
The inhibition of polyomavirus DNA replication in SMC1 knockin mutant cells suggests an important role for SMC1 phosphorylation in polyomavirus replication. Moreover, the fact that the inhibition is more striking in SMC1S957AS966A than in ATM–/– cells suggests the possibility of an additional pathway such as the ATR pathway for SMC1 phosphorylation in virus-infected cells. Consistent with this is the finding that low-dose UV radiation induces the phosphorylation of SMC1, presumably by ATR (18). Further investigations focusing directly on SMC1 and its role in viral DNA replication will be needed to confirm and extend the present observations.
ACKNOWLEDGMENTS
We thank Philip Leder (Harvard Medical School, Boston, MA) for providing the ATM+/+ p53–/– and ATM–/– p53–/– mouse embryo fibroblasts and Michael Kastan (St. Jude Children's Research Hospital, Memphis, TN) for providing the SMC1WT and SMC1S957AS966A cells. We also thank Jim DeCaprio and Matthew Weitzman for their advice and John Carroll and Lori Cobb-Kreisman for their technical assistance.
This work was supported by grant RO1 CA90992. T.L.B. is a Virginia and D. K. Ludwig Professor of Cancer Research and Teaching.
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