ABSTRACT
Adenovirus (Ad) mutants that lack early region 4 (E4) activate the phosphorylation of cellular DNA damage response proteins. In wild-type Ad type 5 (Ad5) infections, E1b and E4 proteins target the cellular DNA repair protein Mre11 for redistribution and degradation, thereby interfering with its ability to activate phosphorylation cascades important during DNA repair. The characteristics of Ad infection that activate cellular DNA repair processes are not yet well understood. We investigated the activation of DNA damage responses by a replication-defective Ad vector (AdRSVβgal) that lacks E1 and fails to produce the immediate-early E1a protein. E1a is important for activating early gene expression from the other viral early transcription units, including E4. AdRSVβgal can deliver its genome to the cell, but it is subsequently deficient for viral early gene expression and DNA replication. We studied the ability of AdRSVβgal-infected cells to induce cellular DNA damage responses. AdRSVβgal infection does activate formation of foci containing the Mdc1 protein. However, AdRSVβgal fails to activate phosphorylation of the damage response proteins Nbs1 and Chk1. We found that viral DNA replication is important for Nbs1 phosphorylation, suggesting that this step in the viral life cycle may provide an important trigger for activating at least some DNA repair proteins.
INTRODUCTION
Ad contains a 36-kbp double-stranded linear DNA genome. The protein products of early region 4 (E4) are important for modulating splicing, apoptosis, transcription, DNA replication, and DNA repair pathways (reviewed in references 41 and 48). Infection with E4 mutants induces a cellular DNA damage response (DDR) that involves the activation of DNA repair kinases ataxia telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) (11), which are critical for mediating responses to DNA damage. Cells have evolved an elaborate network of sensor, transducer, and effector proteins that coordinate cell cycle progression with the repair of DNA damage (reviewed in reference 22). Autophosphorylation and activation of the ATM kinase is one of the earliest characterized events in response to double-strand breaks (DSBs). Autophosphorylation of ATM at serine 1981 leads to dimer dissociation, and it has been proposed that this leads to the release of active ATM monomers that phosphorylate downstream effector molecules such as the protein product of the gene responsible for Nijmegen breakage syndrome (Nbs1), 53BP1, Chk2, histone H2AX, mediator of DNA damage checkpoint protein 1 (Mdc1), and BRCA1 (4, 27). The Mre11/Rad50/Nbs1 (MRN) complex is important for ATM activation and phosphorylation of a number of proteins involved in DNA repair and checkpoint signaling (29). ATM autophosphorylation and downstream signaling is profoundly impaired in infections with wild-type adenovirus type 5 (Ad5) due to degradation of MRN complex proteins (11), an observation consistent with the idea that the MRN complex functions as a DNA damage sensor that collaborates with transducing kinases to activate DNA repair, cell cycle checkpoint, and apoptosis pathways. The MRN complex also plays an important role in the physical repair of DSBs by providing a scaffold that holds DNA breaks together during ligation and repair (2). Thus, the MRN complex acts as both a sensor and an effector of ATM activation and signaling in response to E4 mutant infections and after the introduction of DNA DSBs (11, 29). ATR is also active following E4 mutant infections. ATR responds to several types of DNA damage, but a common theme is the presence of RPA-coated single-stranded DNA (ssDNA) that is produced during repair of damaged DNA or when replication forks stall at sites of DNA damage (15, 52). Ad DNA replication produces ssDNA intermediates during its replication (43) that could also serve to activate ATR responses.
The cellular DDR induced by E4 mutant infection inhibits viral DNA replication (19, 28, 31, 32) and results in the concatenation of viral genomes (7, 39, 45). Ad has evolved several mechanisms to counteract the detrimental effects of the DDR on its life cycle. E4 produces an 11-kDa protein from open reading frame (ORF) 3 (E4-11kDa) and a 34-kDa protein from ORF 6 (E4-34kDa) that each form a physical complex with DNA-PK, which is a critical enzyme for repair by nonhomologous end-joining and for the production of E4 mutant genome concatemers (7). E4-34kDa forms a complex with the E1b-55kDa protein and interacts with a cellular CUL5-containing E3 ubiquitin ligase (6, 14, 36). This complex targets several DDR proteins for ubiquitination and proteasome-mediated degradation, including Mre11 of the MRN complex (11, 39), ligase IV (3), and the cellular tumor suppressor p53 (36). E4-11kDa causes the redistribution of MRN complex proteins away from sites of active viral DNA replication to nuclear track-like structures (19, 39) and cytoplasmic aggresomes located at the periphery of the nucleus (1).
The features of Ad infection required to induce the cellular DDR are not yet completely understood. Incoming genomes are linear double-stranded DNA (dsDNA) templates with covalently attached 5′ terminal proteins and are associated with the virion core DNA-binding proteins V and VII (37, 44). This template could itself serve as a trigger for activating cellular DDRs (48). Viral early gene expression produces multiple regulatory proteins. The E1a protein has transforming properties that stimulate cells to enter S phase (49) and could potentially contribute to activation of the cellular DDR (18). Ad replicates its DNA by a protein priming and strand displacement mechanism, using its own DNA polymerase (12, 43). The presence of unusual replication intermediates and ssDNA produced during E4 mutant replication could also potentially activate the DDR.
Localization of the DDR protein Mdc1 in discrete foci and phosphorylation of the histone variant H2AX are among the earliest events in the DDR to ionizing radiation (reviewed in reference 17). We observed Mdc1 in foci at early times after infection with either Ad5 or an E4 mutant, prior to the onset of viral DNA replication (31). Nichols et al. (34) found that a replication-defective virus lacking E2 is still able to activate focal accumulations of phosphorylated H2AX (γH2AX), suggesting that some aspects of the DDR do not require viral DNA replication. Karen and Hearing (24) recently found that transcription-mediated remodeling of the Ad genome (13) was critical for activation of ATM phosphorylation, suggesting that the chromatin structure of the incoming viral genome may prevent DDR activation.
We have investigated induction of DDRs by a replication-defective Ad vector, AdRSVβgal, which carries the β-galactosidase gene driven by the Rous sarcoma virus promoter in place of the viral E1 region (40). This vector lacks the E1 genes needed to efficiently activate transcription of the other viral early genes, including the E2 genes that encode viral DNA replication proteins, and the E4 genes responsible for inactivating the cellular DDR. This mutant is thus profoundly defective, both for viral early gene expression and DNA replication, although it does synthesize β-galactosidase from the engineered expression cassette. We found that AdRSVβgal is able to activate redistribution of Mdc1 proteins into foci. Mdc1 focus formation correlates with the multiplicity of infection (MOI), which is consistent with the idea that this protein forms foci in response to incoming viral DNA genomes. Interestingly, replication-defective AdRSVβgal infection does not result in phosphorylation of Nbs1 and Chk1, which are normally phosphorylated by the kinases ATM and/or ATR during the cellular DDR. We see a strong correlation between the ability to replicate viral DNA and phosphorylation of Nbs1 and Chk1. Our results suggest that incoming viral genomes may be sufficient to stimulate some aspects of the cellular DDR, but other repair responses may be activated by the process of viral DNA replication.
MATERIALS AND METHODS
Cells and viruses.HeLa, W162 (46), and HEK293 cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 10 U of penicillin/ml, and 10 μg of streptomycin/ml. Wild-type Ad5 and E4 mutants were propagated on W162 cells, which complement the E4 mutant defect. E4 mutants used in the present study include H5dl1007, H5dl1010, and H5dl1014 (8). The status of the E4 genetic regions encoding E4-11kDa and E4-34kDa in each of the mutants is indicated in Table 1. AdRSVβgal (40) was propagated on HEK293 cells that supply E1 gene products in trans. Ad5 titers were determined on both HEK293 and W162 cells and expressed as fluorescent focus-forming units (FFU)/ml (35). The titers for AdRSVβgal and the E4 mutants were determined on HEK293 and W162 cells, respectively. A conversion factor using Ad5 titers on both HEK293 and W162 cells was calculated and was used to determine the W162 titer equivalent for AdRSVβgal. HeLa cells were infected at different MOIs using W162 titers of Ad5 and the mutants. In some experiments, virion DNA was prepared from aliquots of purified virus stocks and quantified to determine the concentration of viral DNA in the original stock. We used the reported molecular weight of Ad5 DNA of about 2.4 × 107 (42) to calculate the concentration of genomes present in the stock, which was equated to the concentration of virion particles. Titers were then expressed as particles/ml.
Status of theE4-11kDa and E4-34kDa gene E4 mutants used in this study
Immunofluorescence analysis and microscopy.HeLa cells were grown on coverslips in 35-mm dishes. Cells were either uninfected or infected with wild-type Ad5 or the indicated viral mutants. In some cases, the cells were treated with 40 μg of α-amanitin (Sigma-Aldrich)/ml, a RNA polymerase II inhibitor, 30 min after infection to study the role of transcription in Mdc1 focus formation. Cells were fixed at various hours postinfection (hpi) in paraformaldehyde (PFA). In brief, cells were washed in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4), gently fixed with 1% PFA in PBS for 1 min, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and then fixed with 4% PFA for 10 min. The fixed cells were blocked for 30 min in blocking reagent buffer (100 mM Tris [pH 7.5], 150 mM NaCl, 0.5% blocking reagent [Amersham]) and incubated with primary antibodies in blocking reagent buffer. The primary antibodies and the dilutions used for immunostaining were rabbit polyclonal anti-Mdc1 (Bethyl Labs) at 1:200, mouse monoclonal antibody against E2-72kDa (kindly provided by A. Levine) at 1:100, and goat polyclonal anti-Mre11 (Santa Cruz Biotechnology) at 1:400. After three 5-min washes in PBS, the cells were incubated with appropriate secondary antibodies (Invitrogen), including Alexa Fluor 594 donkey anti-rabbit IgG (H+L), Alexa Fluor 488 donkey anti-mouse IgG(H+L), and/or Alexa Fluor 594 donkey anti-goat IgG(H+L) at dilutions of 1:2,000. After three 5-min washes in PBS, the cells on coverslips were mounted on glass slides with Vectashield mounting medium (Vector Laboratories, Inc.). The cells were visualized and scored using the ×100 objective of a Nikon Eclipse E-400 microscope. Images were captured using SPOT Advanced charge-coupled device and capture software (Diagnostic Instruments, Inc.). Representative images were chosen, and composite images were assembled using Adobe Photoshop CS5 software.
Western blotting.HeLa cells were grown in 35-mm dishes. Cells were either uninfected or infected with wild-type Ad5 and mutant viruses. In some cases, the cells were treated with UV (100 mJ/cm2) as indicated. Cells from each 35-mm dish were harvested, washed with ice-cold PBS, and pelleted by centrifugation. Cell pellets were lysed in 200 μl of radioimmunoprecipitation assay lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1.0% Igepal CA-630, 0.5% deoxycholic acid) supplemented with protease inhibitors, aprotinin, and leupeptin at 5 μg/ml (Amresco) and a phosphatase inhibitor cocktail in accordance with the manufacturer's instructions (Sigma-Aldrich). Cell lysates were sonicated, and total protein levels in samples were measured by Bradford assay using Coomassie Plus protein reagent (Pierce), according to the manufacturer's instructions. Samples with equal amounts of total protein (50 to 120 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 5, 8, 10, or 15% polyacrylamide gels. The separated proteins were transferred overnight to enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham). The membranes were probed with specific primary antibodies diluted either in 5% nonfat dry milk or 5% bovine serum albumin (BSA) dissolved in 1× Tris-buffered saline (0.02 M Tris-HCl [pH 7.4], 0.136 M NaCl, 0.1% Tween 20) (TBST). The primary antibodies used in immunoblotting were mouse monoclonal antibody against E2-72kDa (kindly supplied by A. Levine) at 1:1,000 dilution, goat polyclonal anti-Mre11 antibody (Santa Cruz Biotechnology) at 1:200, rabbit polyclonal anti-E4-11kDa antibody (a gift from G. Ketner) at 1:700, rabbit monoclonal antibody against Chk1 phosphoserine 345 at 1:1,000, mouse monoclonal antibody against Chk1 (Cell Signaling) at 1:1,000, rabbit polyclonal antibody against Nbs1 phosphoserine 343 (Santa Cruz Biotechnology) at 1:200, and goat polyclonal antibody against Nbs1 (Santa Cruz Biotechnology) at 1:200. After three 15-min washes in 1× TBST, the protein blots were incubated with horseradish peroxidase-conjugated anti-goat (Santa Cruz Biotechnology) and anti-rabbit and anti-mouse (Amersham) secondary antibodies diluted 1:1,500 in 5% nonfat dry milk or 5% BSA. After four 15-min washes in 1× TBST, the membranes were incubated with ECL reagent (Amersham) to generate chemiluminescence signals, which were subsequently captured on ECL Hyperfilm (Amersham). For the quantitation of protein levels, the membranes were probed with alkaline phosphatase-conjugated anti-goat, anti-rabbit, and anti-mouse secondary antibodies (Santa Cruz Biotechnology) at a 1:1,500 dilution. Protein blots were subsequently incubated with enhanced chemifluorescence substrate (Amersham), and images were captured using a Storm 860 phosphorimager (Molecular Dynamics). The proteins were quantified from captured images using ImageQuant 5.2 (Molecular Dynamics) software.
Viral DNA analysis.Total and nuclear DNA was isolated from uninfected and infected cells as described previously (47). In brief, cells were washed twice with ice-cold PBS and subsequently harvested and centrifuged to recover cell pellets. For total DNA, cell pellets were lysed in 0.05 M Tris [pH 7.8], 0.0025 M EDTA, and 0.25% SDS containing proteinase K at 0.4 mg/ml. DNA was extracted twice with phenol and chloroform and precipitated with ethanol, and the recovered DNA was dissolved in nuclease-free water. For the isolation of nuclear DNA, cell pellets were processed through two rounds of gentle resuspension in lysis buffer (0.14 M NaCl, 1.5 mM MgCl2, 100 mM Tris [pH 8.6], 0.5% Igepal CA-630, 1 mM dithiothreitol) to solubilize cytoplasmic membranes, followed by centrifugation to recover the nuclei. The pelleted nuclei were used to prepare total DNA as described above.
For dot blot analysis, total and nuclear DNA samples were treated with RNase (DNase-free) for 1 h at 0.5 μg/ml. Samples were extracted with phenol and chloroform, and DNA was precipitated using ethanol. RNase-free DNA samples (2 to 10 μg) were denatured by boiling at 100°C and subsequently chilled on ice. DNA samples were adjusted to a final concentration of 6× SSC using 20× SSC (3 M NaCl, 300 mM sodium citrate). The dot blot manifold was set up with 6× SSC presoaked Hybond-N nylon membrane (Amersham) for DNA transfer. DNA samples in 6× SSC were spot transferred to a Hybond-N nylon membrane through the wells of a dot blot manifold under gentle vacuum. DNA was fixed to the membrane by UV cross-linking using a UV transilluminator. The membranes were probed with a radioactive Ad specific probe as described below for Southern blot analysis.
For Southern blot analysis, 10 μg of total DNA from each sample was digested with EcoRI and subjected to electrophoresis in a 1% agarose gel for 20 h at 20 V. DNA was transferred to Hybond-N nylon membrane (Amersham), which was subsequently hybridized with Ad-specific probe. Ad-specific 32P probe was synthesized using Ad5 genomic DNA as a template for the Multiprime DNA labeling system (GE Healthcare/Amersham) in accordance with the manufacturer's instructions. Hybridization with 5 × 106 cpm of probe/ml was performed at 65°C for 20 h. The membranes were subjected to phosphorimaging analysis, and the DNA levels were quantified using ImageQuant 5.2 (Molecular Dynamics) software.
RESULTS
Mdc1 focus formation is correlated with the MOI and requires viral genome transcription but not replication.Relocalization of Mdc1 into foci is one of the earliest events observed in activation of the DDR in response to ionizing radiation (17). We have previously found that Mdc1 focus formation can be observed by 4 hpi before the onset of viral DNA replication in both Ad5 and E4 mutant infections (31). AdRSVβgal lacks E1 and is therefore deficient for both viral early gene expression and DNA replication (5). We investigated the distribution of Mdc1 in cells infected with replication-defective AdRSVβgal in comparison to replication-competent wild-type Ad5, and E4 mutant H5dl1007, which replicates its DNA similarly to Ad5 at MOI 30 FFU/cell, to determine whether Mdc1 focus formation is efficient in replication-defective infections. Immunofluorescence micrographs showing Mdc1 redistribution into foci in cells infected with the indicated viruses are shown in Fig. 1A. Efficient Mdc1 focus formation was seen in all of the infections. We infected cells at MOIs of 30 and 300 FFU/cell to determine whether there is a correlation between Mdc1 focus formation and the levels of input virus. Uninfected cells showed a background level of Mdc1 focus formation of ca. 7%. Mdc1 focus formation was observed in 15 to 20% of the cells infected at MOI 30 FFU/cell with all three viruses tested; this was an increase of at least 2-fold over background (Fig. 1B, top graph). Mdc1 focus formation was observed in 35 to 50% of the cells infected at an MOI of 300 FFU/cell (Fig. 1B, bottom graph). This 6- to 7-fold increase in Mdc1 focus formation over background was observed with each of the viruses tested. These results indicate that the efficiency of Mdc1 focus formation was correlated with levels of virus used to infect the cells and did not depend on viral DNA replication. These results are consistent with the idea that the delivery of viral genomes to the cell by infection is sufficient to induce this aspect of the DDR response even in the absence of viral DNA replication.
Mdc1 focus formation in Ad-infected cells is correlated with MOI. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1−) at an MOI of 30 or 300 FFU/cell as indicated. The cells were then fixed with paraformaldehyde at 5 hpi and immunostained with antibody against Mdc1. (A) Immunofluorescence micrographs showing Mdc1 redistribution into large foci in cells infected with the indicated viruses at an MOI of 30 FFU/cell. (B) Cell cultures were infected with the indicated viruses, blindly scored for Mdc1 foci, and the percentages of cells with large Mdc1 foci were plotted for infections performed at an MOI of 30 or 300 FFU/cell. Error bars show the standard errors of the mean from three independent experiments.
To confirm that AdRSVβgal infections were indeed defective for early gene expression and DNA replication, HeLa cells were infected at an MOI of 30 FFU/cell, and early gene expression was assayed by Western blotting with an antibody that detects the viral 72-kDa DNA-binding protein produced from E2 (E2-72kDa) and the E4-11kDa protein produced from E4 open reading frame 3 (ORF3). Figure 2A shows that E2-72kDa is efficiently produced in Ad5 and H5dl1007 infections but is dramatically reduced in AdRSVβgal infections, confirming that AdRSVβgal is deficient for expression of this protein. E4-11kDa protein levels were reduced in AdRSVβgal infections compared to Ad5, but not as dramatically as the E2-72kDa protein (Fig. 2B). These results indicate that AdRSVβgal has leaky expression of E4-11kDa even in the absence of functional E1. We next assessed the ability of AdRSVβgal to replicate its DNA to determine whether the reductions in early gene expression were sufficient to prevent viral DNA replication under our infection conditions. HeLa cells were infected with AdRSVβgal at an MOI of 30 or 300 FFU/cell, and total DNA levels at 4 and 24 hpi were measured by dot blot analysis (Fig. 2C). We saw no increase in viral DNA levels between 4 and 24 hpi at either MOI, confirming that AdRSVβgal is profoundly replication defective. We also measured the levels of total and nuclear DNA in Ad5, H5dl1007, and AdRSVβgal infections at 4 hpi to confirm that the mutant viruses can efficiently deliver their DNA genomes to the nucleus. The levels of total and nuclear DNA were similar for all of the infections, and AdRSVβgal was not deficient for the delivery of viral DNA to the nucleus compared to Ad5 (Fig. 2D).
AdRSVβgal is defective for viral early gene expression and viral DNA replication. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1−) at an MOI of 30 FFU/cell. (A and B) Western blotting was performed on extracts prepared at 24 hpi with antibody against the E2-72kDa (72K) (A) and E4-11kDa (11K) (B). (C) Total DNA samples from cells infected with AdRSVβgal at an MOI of 30 or 300 FFU/cell were prepared at 4 and 24 hpi and analyzed by dot blotting. DNA levels were expressed as the fold difference from AdRSVβgal DNA levels measured at 4 hpi for an MOI of 30 FFU/cell, which was set at 1. (D) HeLa cells were infected with the indicated viruses at an MOI of 30 FFU/cell. Total (T) and nuclear (N) DNA samples were prepared at 4 hpi (prior to the onset of DNA replication) and analyzed by dot blotting. DNA levels were expressed relative to the Ad5 levels for both total and nuclear DNA, which were each set at 1. Error bars show the standard errors of the mean from three independent experiments.
AdRSVβgal has been engineered to produce β-galactosidase driven by a Rous sarcoma virus promoter, which is expected to be constitutively active. Since recent results from Karen and Hearing (24) suggest that transcription-mediated remodeling of the viral chromatin is needed to activate phosphorylated ATM focus formation, we next sought to determine whether active transcription was required for Mdc1 focus formation in infected cells. HeLa cells were infected with Ad5, AdRSVβgal, or H5dl1007 and immediately treated with α-amanitin to inhibit transcription from the incoming genomes. Inhibiting transcription significantly decreased Mdc1 focus formation in all infections (Fig. 3A and B). In control experiments, we found that α-amanitin treatment from 30 min to 10 hpi was sufficient to block E2-72kDa production in Ad5 infections throughout the length of the early phase (Fig. 3C), indicating that the treatment effectively blocked early gene expression. These results suggest that transcription from the viral genome is important for the cell's ability to respond to infection by forming Mdc1 foci.
Mdc1 focus formation depends on transcription from the viral genome. (A) HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1−) viruses at an MOI of 30 FFU/cell. At 30 min postinfection, experimental cells were left untreated or treated with 40 μg of α-amanitin/ml (α-am). The cells were fixed with paraformaldehyde at 5 hpi and immunostained with antibody against Mdc1. (B) Cells in each infected culture were blindly scored for Mdc1 focus formation in three independent experiments, and the results are graphed. Error bars show the standard errors of the mean. (C) As a control, we measured the effect of α-amanitin treatment on Ad5 early gene expression. Ad5-infected HeLa cells were left untreated or treated with α-amanitin from 30 min postinfection. Protein extracts prepared at 10 hpi were subjected to Western blot analysis with an antibody that detect E2-72kDa (72K) as a marker for early gene expression.
AdRSVβgal infection fails to activate phosphorylation of Nbs1 and Chk1 and redistributes Mre11 to nuclear tracks.Since AdRSVβgal infection induces Mdc1 focus formation (Fig. 1), which is an early DDR, we next investigated its ability to induce phosphorylation of Nbs1 and Chk1. In E4 mutant infections Nbs1 and Chk1 represent examples of several substrates that are phosphorylated by the ATM and/or ATR kinases following sensing of DNA damage by the MRN complex (11). HeLa cells were either left uninfected or infected with Ad5 or the indicated mutants at an MOI of 30 FFU/cell and then cultured for 22 to 24 hpi. Protein extracts prepared from these cultures were subjected to SDS-PAGE and Western blot analysis with antibodies against phosphorylated Nbs1 or phosphorylated Chk1 (Fig. 4). Ad5 does not efficiently phosphorylate Nbs1 or Chk1 as expected, since its E4 proteins inactivate Mre11 (11). The E4 deletion mutant H5dl1007 induced substantial levels of Nbs1 and Chk1 phosphorylation. In contrast, AdRSVβgal did not activate phosphorylation of either protein. The levels of unphosphorylated Nbs1 and Chk1 were not significantly affected by infection with these viruses, with the exception of unphosphorylated Nbs1 in Ad5 infections, which was reduced compared to uninfected controls (Fig. 4) due to the degradation of MRN complex proteins by Ad5 E4 proteins, as shown previously (11). Our results indicate that although AdRSVβgal infection is sufficient to stimulate Mdc1 focus formation (Fig. 1), it does not activate a full DDR that includes phosphorylation of Nbs1 and Chk1.
Replication-defective AdRSVβgal fails to activate phosphorylation of Nbs1 and Chk1. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, or AdRSVβgal (E1−) at an MOI of 30 FFU/cell for 22 to 24 h. Protein extracts prepared from these cultures were subjected to SDS-PAGE and Western blot analyses using antibodies against phosphorylated Nbs1 (pNbs1) (A) or phosphorylated Chk1 (pChk1) (B). Extracts from the same infections were assayed in Western blots with antibodies to detect unphosphorylated epitopes of Nbs1 and Chk1, and actin (middle and lower panels, respectively).
We next sought to determine whether Mre11 is affected by AdRSVβgal infections. Ad5 produces E4 proteins that inactivate Mre11 by degradation and redistribution, thereby preventing the activation of many aspects of the cellular DDR. AdRSVβgal lacks E1b and therefore cannot make the E1b-55kDa/E4-34kDa complex that targets Mre11 for proteasome-mediated degradation. However, AdRSVβgal does express E4-11kDa, albeit at reduced levels compared to Ad5 (Fig. 2B), and could therefore affect the distribution of Mre11. We measured Mre11 levels and its distribution in AdRSVβgal infections. HeLa cells were either uninfected or infected with Ad5, H5dl1007, or AdRSVβgal at an MOI of 30 FFU/cell. Western blotting with Mre11 antibody was performed using extracts prepared at 24 hpi, and the results are shown in Fig. 5A. Mre11 is reduced in Ad5 infections as expected, but the levels of Mre11 are relatively unaffected by infection with either H5dl1007 or AdRSVβgal. We next assessed Mre11 localization by immunofluorescence staining of infected cells with antibodies against Mre11 and the viral E2-72kDa DNA-binding protein (Fig. 5B). Mre11 was substantially degraded in Ad5-infected cells. In H5dl1007-infected cells, Mre11 colocalized with viral DNA replication centers containing E2-72kDa, as shown previously (39). In AdRSVβgal infections Mre11 is detected in nuclear tracks. The appearance of Mre11 in tracks in AdRSVβgal infections is delayed relative to Ad5 in time course experiments (data not shown), but the cells with Mre11 redistributed to nuclear tracks accumulate steadily and by 20 hpi represent the majority of the infected cells. This result indicates that AdRSVβgal infection is able to redistribute Mre11 into nuclear tracks, which could potentially impact its ability to activate Nbs1 and Chk1 phosphorylation.
Redistribution of Mre11 to nuclear tracks is not sufficient to prevent Nbs1 phosphorylation. HeLa cells were either uninfected (UI) or infected with Ad5, H5dl1007, H5dl1010, or AdRSVβgal (E1−) viruses at an MOI of 30 FFU/cell. (A) Western blotting was performed on extracts prepared at 24 hpi from the indicated infections, using antibody against Mre11 (top panel). Phosphorimaging analysis of three independent blots was performed, and the results were plotted (bottom panel). Error bars show the standard error of the mean. (B) Mre11 localization was determined by double immunofluorescence staining of cells infected with the indicated viruses and fixed at 20 to 24 hpi and immunostained with Mre11 and E2-72kDa antibodies. (C) Western blotting was performed on extracts prepared at 24 hpi from the indicated infections, using antibody against phosphorylated Nbs1 (pNbs1) (top panel) or phosphorylated Chk1 (pChk1) (bottom panel).
E4-11kDa-mediated redistribution of Mre11 to nuclear tracks is not sufficient to prevent Nbs1 phosphorylation.E4-11kDa-mediated distribution of Mre11 to nuclear tracks affects the ability of ATR to phosphorylate Chk1 but does not prevent the activation of ATM in infections with viruses that express E4-11kDa but lack the ability to degrade the MRN complex (10). Nbs1 is potentially a substrate of either the ATM or ATR kinase. We performed infections with E4 mutant H5dl1010 to address the role of Mre11 redistribution in Nbs1 phosphorylation. H5dl1010 lacks an intact gene for E4-34kDa and is unable to degrade Mre11 (data not shown) but expresses E4-11kDa and therefore efficiently redistributes Mre11 to nuclear tracks (Fig. 5B). Western blotting indicated that H5dl1010 did not activate Chk1 phosphorylation, as expected (10). However, Nbs1 was phosphorylated in H5dl1010 infections (Fig. 5C) despite efficient Mre11 redistribution (Fig. 5B). This result is consistent with the idea that E4-11kDa-mediated Mre11 redistribution does not prevent ATM activation (10) and suggests that ATM-mediated activation of Nbs1 occurs in H5dl1010 infections. Although H5dl1010 induction of Nbs1 phosphorylation was not as strong as in H5dl1007 infections, this is likely because ATR cannot contribute to Nbs1 phosphorylation in H5dl1010 infections. A further implication of this result is that the failure of AdRSVβgal to activate Nbs1 phosphorylation is not simply due to E4-11kDa-mediated Mre11 redistribution, since AdRSVβgal redistributes Mre11 similarly to H5dl1010 (Fig. 5B) but does not activate Nbs1 phosphorylation (Fig. 5C).
DNA damage triggers can still induce Nbs1 phosphorylation in AdRSVβgal-infected cells.The failure of AdRSVβgal to activate Nbs1 phosphorylation led us to hypothesize that this replication-defective virus might be missing an activation trigger that is provided in H5dl1010 infections. An alternative possibility is that AdRSVβgal infection inactivates the DDRs needed for Nbs1 phosphorylation. To address these possibilities, we exposed AdRSVβgal-infected cells to UV light to see whether these cells were still capable of responding to a known DNA damage trigger. UV treatment induces both ATM (50) and ATR (15) responses, and cells that lack functional ATM are defective for repair of UV-induced DNA damage (21). HeLa cells were either uninfected or infected with Ad5 or AdRSVβgal at an MOI of 30 FFU/cell and, at 22 hpi, were either exposed to UV (100 mJ/cm2) or left untreated. We harvested the cultures at 24 hpi, and protein lysates were analyzed by Western blotting with antibody against phosphorylated Nbs1 (Fig. 6). Phosphorylated Nbs1 levels increased in AdRSVβgal-infected cells treated with UV compared to untreated infections. Ad5-infected cells with or without UV treatment did not show any appreciable difference in phosphorylated Nbs1 levels. These results indicate that while Ad5 is capable of preventing UV-induced Nbs1 phosphorylation, presumably through its inactivation of Mre11 by degradation, AdRSVβgal is not. Our data suggest that AdRSVβgal-infected cells are capable of phosphorylating Nbs1 in response to a UV DNA damage trigger and support the idea that AdRSVβgal infection by itself fails to provide an activating trigger needed to induce efficient Nbs1 phosphorylation.
UV treatment of AdRSVβgal-infected cells activates the phosphorylation of Nbs1. HeLa cells were either left uninfected (UI) or infected with Ad5 or AdRSVβgal (E1−) at an MOI of 30 FFU/cell, and after 22 h the cultures were exposed to UV (100 mJ/cm2) as indicated or left untreated. Cultures were harvested at 24 hpi, and protein lysates were prepared and analyzed by Western blotting with antibody against phosphorylated Nbs1 (pNbs1). A representative Western blot is shown in panel A. Phosphorimaging analysis of three independent blots was performed, and a graph of the results is shown in panel B. Error bars show the standard errors of the mean.
Viral DNA replication is important for activating Nbs1 phosphorylation.AdRSVβgal is replication defective and fails to induce phosphorylation of Nbs1 or Chk1, whereas H5dl1010, which replicates its DNA normally at a high MOI (8), activated Nbs1 phosphorylation (Fig. 5). We next explored the possibility that viral DNA replication may provide a trigger that is important for inducing these responses. E4 mutant H5dl1007 replicates its DNA similarly to wild-type Ad5 at a high MOI (8), but at lower MOIs E4 mutants that lack the genes for both E4-34kDa and E4-11kDa have substantial DNA replication defects (20, 47). We investigated the induction of Nbs1 phosphorylation following low-multiplicity infections by H5dl1007 to determine whether viral DNA replication was correlated with inducing Nbs1 phosphorylation. HeLa cells were infected with wild-type Ad5 at an MOI of 30 FFU/cell or H5dl1007 at MOIs of 30, 3, 1, and 0.3 FFU/cell. Even at the lowest MOI tested with H5dl1007, >70% of the cells were infected, as determined by immunofluorescence detection of E2-72kDa at 24 hpi (data not shown). At the low MOIs of 1 and 0.3 FFU/cell, E2-72kDa staining was primarily diffuse and replication foci containing this protein were rarely observed (data not shown). Total DNA was isolated from infected HeLa cells at 22 to 24 hpi for Southern blot analysis (Fig. 7A, top panel). The levels of viral DNA were quantified by phosphorimaging analysis (Fig. 7A, bottom graph). The H5dl1007 DNA levels were similar to Ad5 at an MOI of 30 FFU/cell and 30-fold lower at an MOI of 3 FFU/cell and were not detectable above the background signal of uninfected HeLa cells at an MOI of 1 or 0.3 FFU/cell. We next measured the levels of phosphorylated Nbs1 to assess the activation of cellular DDR proteins in H5dl1007 infections performed at different MOIs. Cell extracts were prepared at 22 hpi for Western blot analysis with antibody against phosphorylated Nbs1. The results are shown in Fig. 7B. H5dl1007 infection performed at an MOI of 30 or 3 FFU/cell resulted in a substantial increase in the levels of phosphorylated Nbs1 compared to the uninfected HeLa cell control. In contrast, H5dl1007 infections carried out at an MOI of 0.3 FFU/cell did not activate Nbs1 phosphorylation.
Viral DNA replication is important for activating Nbs1 phosphorylation. HeLa cells were either uninfected (UI) or infected with wild-type Ad5, AdRSVβgal (E1-), H5dl1007, or H5dl1014 at the indicated MOIs and cultured for 22 to 24 hpi. The MOIs used are in parentheses in FFU/cell. (A) Southern blot analysis was performed with 10 μg of EcoRI-digested DNA prepared from each infection. The EcoRI C fragment used for comparison of viral DNA levels is shown in the top panel. Viral DNA levels were quantified by phosphorimaging analysis (lower graph). E4 mutant H5dl1007 DNA levels are expressed as the fraction of the Ad5 level, which was set at 1. Error bars indicate the standard deviations from three independent experiments. (B) Protein extracts from infected cells were subjected to SDS-PAGE and Western blot analysis with antibody against phosphorylated Nbs1. (C) Cells were infected with AdRSVβgal (E1−) and H5dl1007 at MOIs of 30 and 3 FFU/cell, respectively. DNA levels from the indicated infections and times (hpi) were measured by dot blot analysis and are expressed as the fraction of AdRSVβgal DNA levels measured at 4 hpi, which was set at 1. Error bars indicate the standard deviations from three independent experiments. (D) Total DNA samples were prepared from the indicated infections and hpi, and the samples were analyzed by dot blotting. DNA levels were expressed as the fold difference from H5dl1007 DNA levels measured at 4 hpi after infection with 200 particles/cell (top graph) or 800 particles/cell (bottom graph), which was set at 1. Error bars show the standard errors of the mean from three independent experiments. (E) Protein extracts from the indicated infections were subjected to SDS-PAGE and Western blot analysis with antibody against phosphorylated Nbs1 (pNbs1). In panels D and E, MOIs were expressed as virion particles/cell (see Materials and Methods). UV-treated uninfected cells were used as a positive control for induction of pNbs1.
We next addressed the possibility that a certain threshold of viral DNA ends might be needed to activate a DDR and that, when lower levels of virus are used for infection, replication is required to produce this threshold number of ends. We performed dot blot analysis to measure the levels of viral DNA present in cells infected with replication-defective AdRSVβgal at an MOI of 30 FFU/cell or with H5dl1007 at an MOI of 3.0 FFU/cell to address this issue. DNA samples were prepared at 4 and 22 hpi to measure viral DNA levels before and after the typical onset of viral replication, which occurs at about 12 hpi (Fig. 8). We observed a modest increase in H5dl1007 DNA levels of 3- and 4-fold between 4 and 22 hpi at an MOI of 3.0 FFU/cell (Fig. 7C), and H5dl1007 induced the phosphorylation of Nbs1 at this MOI (Fig. 7B). In contrast, input AdRSVβgal DNA levels at 4 hpi at an MOI of 30 FFU/cell were already higher than the level achieved by H5dl1007 replication for 22 h in cells infected at 3.0 FFU/cell. AdRSVβgal DNA levels were actually less at 22 hpi than at 4 hpi, confirming that no DNA replication occurred during AdRSVβgal infections at this MOI (see also Fig. 2C). AdRSVβgal did not induce phosphorylation of Nbs1 at an MOI of 30 FFU/cell (Fig. 7B). The observation that H5dl1007 induces a damage response at a low MOI despite achieving fewer DNA templates following replication than were present following infection with AdRSVβgal at a high MOI argues that achieving a threshold level of viral DNA is not sufficient to activate Nbs1 phosphorylation.
E4 mutant-induced phosphorylation of Nbs1 and Chk1 correlates with the onset of viral DNA replication. HeLa cells were infected with H5dl1007 at 30 FFU/cell. Total DNA was isolated at 0, 4, 8, 16, and 22 hpi for Southern blot analysis and DNA levels were quantified by phosphorimaging analysis. (A) E4 mutant DNA levels were measured as the fold increase over the background level detected in samples prepared from uninfected cells, which was set at 1. In a parallel experiment, protein lysates were prepared from H5dl1007-infected cells at the times indicated and processed for Western blot analyses with antibodies against phosphorylated Nbs1 (B) and Chk1 (C). Protein levels were quantified by phosphorimaging analysis. The levels of phosphorylated Nbs1 (pNbs1) and Chk1 (pChk1) are expressed as the fold increase compared to the level detected in uninfected HeLa cells, which was set at 1.
AdRSVβgal lacks E1, raising the possibility that its failure to activate some DDRs could be due to its failure to express E1 genes. We addressed this by studying E4 mutant H5dl1014, which has a more substantial DNA replication defect than H5dl1007 (9) but still carries an intact E1 region. H5dl1014 is derived from wild-type Ad5 and carries two deletions that destroy all of the E4 ORFs except ORF4 (8). HeLa cells were infected with H5dl1007 or H5dl1014 at an MOI of 200 or 800 particles/cell. Total DNA was prepared at 4 and 24 hpi and analyzed by dot blotting (Fig. 7D). At an MOI of 200 particles/cell (top panel) H5dl1014 and H5dl1007 had similar levels of input DNA at 4 hpi, which is before the onset of DNA replication (the ratio of H5dl1014/H5dl1007 DNA levels was ∼1.1). This indicates that H5dl1014 and H5dl1007 infections delivered equal amounts of viral DNA to the cell. H5dl1007 DNA levels increased 27-fold, whereas H5dl1014 DNA levels increased only 2.7-fold, by 24 hpi. In parallel infections total cell extracts were prepared at 24 hpi for Western blot analysis with an antibody against phosphorylated Nbs1. Extract prepared from UV-treated cells was used as a control for Nbs1 activation. At 200 particles/cell, H5dl1014 did not activate Nbs1 phosphorylation, whereas H5dl1007 showed a substantial increase in phosphorylated Nbs1 levels compared to the uninfected control (Fig. 7E, top panel). Since these viruses have identical E1 regions, the major difference between the two infections is deficient DNA replication by H5dl1014. When similar experiments were performed using 800 particles/cell, we saw significant increases in viral DNA levels between 4 and 24 hpi (Fig. 7D, bottom panel) and activation of Nbs1 phosphorylation (Fig. 7E, bottom panel) in both H5dl1014 and H5dl1007 infections. H5dl1014 and H5dl1007 showed 22- and 165-fold increases in DNA levels, respectively, at this MOI. Taken together, these results suggest that replication of viral DNA provides a trigger that induces phosphorylation of the DDR protein Nbs1.
Our results indicate that phosphorylation of Nbs1 occurs only in E4 mutant infections capable of replicating viral DNA. Therefore, we next wanted to investigate whether the onset of H5dl1007 DNA replication correlates with the activation of host DDR proteins in time course experiments. HeLa cells were infected with H5dl1007 at 30 FFU/cell. Total DNA was isolated at 0, 4, 8, 16, and 22 hpi for Southern blot analysis. Figure 8A shows the findings from a phosphorimaging analysis of E4 mutant viral DNA levels expressed as the fold increase over the background levels seen in uninfected controls. We found that H5dl1007 DNA levels increased by 10-fold between 12 and 16 hpi, indicating the onset of viral DNA replication. In a parallel experiment, total cell extracts were prepared from H5dl1007-infected cells at the times indicated for Western blot analysis with antibodies against phosphorylated Nbs1 and Chk1. The levels of phosphorylated Nbs1 and Chk1 were similar to the background levels seen in uninfected cells (0 hpi) until ∼12 hpi and then increased by 8- to 10-fold between 12 and 16 hpi (Fig. 8B and C). This correlates very well with the onset of viral DNA replication (Fig. 8A), indicating that viral DNA replication coincides with phosphorylation of the DDR proteins Nbs1 and Chk1.
DISCUSSION
The induction of cellular DDRs has been extensively investigated. DSBs produced by ionizing radiation are sensed by the MRN complex, which activates kinases critical for both cell cycle checkpoint activation and for repair of the broken DNA (22, 29). Ad infection also induces cellular DDRs (11). Ad infection and replication presents the cell with both unusual protein-linked DNA ends and ssDNA replication intermediates that could be interpreted by the cell as signs of DNA damage. We have used a replication-defective Ad vector, AdRSVβgal, to study the activation of DNA repair responses during infections that efficiently deliver Ad genomes to the nucleus but are then incapable of carrying out viral DNA replication (Fig. 2). To our surprise, although AdRSVβgal infection induced Mdc1 focus formation (Fig. 1), it was deficient for the activation of other aspects of the DDR, including the phosphorylation of Nbs1 and Chk1 (Fig. 4).
Mdc1 is one of the earliest proteins to be recruited to the site of DNA breaks induced by ionizing radiation. We have previously observed that the MRN sensor complex is needed for efficient Mdc1 focus formation in Ad-infected cells (31) and that both Mre11 and Mdc1 are bound to viral DNA at early times after infection in chromatin immunoprecipitation (ChIP) experiments (32). Mdc1 focus formation is correlated with MOI (Fig. 1), indicating that it could be a response to the increase in incoming genomes. Ad genomes are packaged in the virion with the core proteins pV and pVII (16, 33, 44). The viral genome bound to core proteins and covalently linked terminal proteins is then delivered to the nucleus during infection (23, 38). ChIP experiments indicate that the Ad genome interacts with histones and that the genomic template for early transcription contains a mix of cellular histones and the viral core protein pVII. During the process of early transcription the genome undergoes chromatin remodeling and pVII is replaced by acetylated histones (26). Karen and Hearing (24) have studied the formation of foci containing phosphorylated ATM (pATM) in E4 mutant infections, and find that pATM colocalizes with Ad genomes that no longer contain the viral core DNA-binding protein pVII. Furthermore, pATM foci are reduced in cells treated with a transcription inhibitor and after infection by Ad E1 mutants that fail to activate any of the viral genome's transcription units. These observations indicated that early transcription-mediated uncoating of pVII from the viral genome is important for pATM focus formation. Likewise, we found that transcription inhibition can also interfere with Mdc1 focus formation in Ad5, E4 mutant, and AdRSVβgal infections (Fig. 3). Although we cannot rule out the possibility that early gene expression per se is required for activating Mdc1 focus formation, the observation that AdRSVβgal induces efficient Mdc1 focus formation (Fig. 1) despite the absence of E1 genes and poor activation of other early genes (Fig. 2), argues against this. Rather, we consider it more likely that transcription-mediated chromatin remodeling is important for Mdc1 focus formation, similar to the pATM foci identified by Karen and Hearing (24). It is possible that the ability of the MRN complex to sense and respond to incoming viral DNA genomes depends on their chromatin configuration and that the exchange of the viral core proteins for a more cell-like chromatin configuration involving cellular histones is critical for the MRN complex to recognize the viral genome as “DNA damage” and activate responses such as Mdc1 and pATM focus formation.
Although AdRSVβgal was able to activate Mdc1 focus formation, which is an early DDR event during Ad infection, we found that it was unable to trigger phosphorylation of either Nbs1 or Chk1 (Fig. 4). Typically, these proteins are substrates of the ATM and/or ATR kinases and are phosphorylated following ionizing radiation. Recently, Carson et al. (10) have shown that redistribution of the MRN complex to nuclear tracks by the E4-11kDa protein can prevent ATR-mediated signaling, including phosphorylation of Chk1. AdRSVβgal can produce E4-11kDa (Fig. 2) and redistribute Mre11 to nuclear tracks (Fig. 5), so it is therefore possible that this explains its failure to activate Chk1. This possibility is supported by the observation that H5dl1010 infections also fail to phosphorylate Chk1 (Fig. 5C). H5dl1010 carries a deletion affecting E4-34kDa and, like AdRSVβgal, does not degrade Mre11 (data not shown) but efficiently redistributes it to nuclear tracks (Fig. 5B). However, H5dl1010 induced phosphorylation of Nbs1, whereas AdRSVβgal did not (Fig. 5C). This raises the possibility that either AdRSVβgal fails to phosphorylate Nbs1 because it has inactivated DDR responses in infected cells or, alternatively, the AdRSVβgal life cycle may be so defective that it fails to produce triggers necessary to activate aspects of the DDR needed for efficient Nbs1 phosphorylation. Interestingly, UV treatment of AdRSVβgal-infected cells induced Nbs1 phosphorylation, although the levels were not quite as high as in UV-treated uninfected cells (Fig. 6). This could be because ATR contributes to Nbs1 phosphorylation in UV-treated uninfected cells, whereas the ATR responses are likely to be inactivated by Mre11 redistribution in AdRSVβgal infections (Fig. 4 and 5) (10). Nevertheless, this result indicates that AdRSVβgal infection does not block the ability of cells to respond to UV-induced DNA damage and suggests that AdRSVβgal infections fail to provide a necessary trigger for activating Nbs1 phosphorylation. We think it is likely that replication-defective infections may also fail to provide triggers needed for activating Chk1 phosphorylation, but this must be confirmed with a vector that lacks E1, as well as the gene encoding the E4-11kDa protein (E4 ORF3), and is therefore not capable of inactivating ATR by Mre11 redistribution to nuclear tracks.
We consistently find that Ad mutant infections that do not result in significant viral DNA replication fail to induce phosphorylation of Nbs1. This includes AdRSVβgal infections, H5dl1007 infections performed at a low MOI, and infections with H5dl1014, which is more defective for DNA replication than H5dl1007 (Fig. 7). We do not find evidence that a threshold level of incoming DNA templates is required to activate Nbs1 phosphorylation (Fig. 7C). Since AdRSVβgal lacks the E1 region in addition to being defective for DNA replication, it is possible that the E1 proteins themselves are important for activating some DDRs (18). However, H5dl1014 and H5dl1007 both carry an intact E1 region, and yet H5dl1014 still failed to activate phosphorylation of Nbs1 under infection conditions that resulted in similar delivery of viral genomes to the cell but very little H5dl1014 DNA replication compared to H5dl1007. When cells were infected with four times more H5dl1014 and H5dl1007, we observed significant DNA replication, as well as efficient Nbs1 phosphorylation in infections with both viruses (Fig. 7D and E). We also found that the phosphorylation of both Nbs1 and Chk1 is coincident with the onset of viral DNA replication in E4 mutant-infected cells in time course experiments (Fig. 8). Our results are most consistent with a model in which Nbs1 and possibly also Chk1 phosphorylation requires replication of input viral DNA. Karen et al. (25) concluded that the input genomes of a replication-defective E4 mutant were sufficient to induce ATM phosphorylation. However, we note that their replication-defective E4 mutant, dl355/inORF3, was actually able to increase DNA levels >100-fold between 8 and 24 hpi in their DNA replication assay. Although this is certainly defective compared to wild-type infections, it is comparable to the 165-fold increase in DNA levels that we observed between 4 and 24 hpi with a similar E4 mutant, H5dl1007, which was sufficient to induce significant Nbs1 phosphorylation (Fig. 7). We suggest that relatively modest levels of viral DNA replication are sufficient for activating DNA replication-dependent DDRs, and while these levels are achieved in typical infections with defective E4 mutants, they are not achieved in AdRSVβgal infections, which show no increase in viral DNA levels between 4 and 24 hpi (Fig. 2 and 7).
Why would viral DNA replication be important for activating cellular DDRs? The ATR kinase is known to respond to ssDNA (51), so it is possible that the ssDNA intermediates produced during Ad replication are an important part of the mechanism involved in triggering ATR activation. Nichols et al. (34) have shown that widespread phosphorylation of the histone H2AX during Ad infection requires viral DNA replication, and their results were consistent with a role for the ATR kinase in this response. ATM is thought to respond to the presence of DSBs (22, 29, 30), and if incoming linear viral DNA genomes mimic such breaks, it is possible that only ATM responses would be active in replication-defective AdRSVβgal infections. However, we have data suggesting that Mdc1 foci still form in cells that lack ATM (D. Gautam and E. Bridge, unpublished data), indicating that this response may be independent of ATM in Ad-infected cells. Preliminary data suggest that while we see significant phosphorylation of ATM in both H5dl1007 and H5dl1010 infections, we see relatively little ATM phosphorylation in AdRSVβgal infections (A. Prakash and E. Bridge, unpublished data). Although Nbs1 is not exclusively a substrate of the ATM kinase, it is noteworthy that we also failed to see Nbs1 phosphorylation in AdRSVβgal infections (Fig. 4). Thus, it is possible that the delivery of viral genomes to the nucleus in the absence of DNA replication is not sufficient for full activation of either ATM or ATR. In vitro studies have shown that ATM activation requires linear dsDNA and the MRN complex (29). Interestingly, these authors also found that the ability of the MRN complex to unwind DNA ends, thereby producing ssDNA, was critical for ATM activation. It is possible that the presence of 5′-terminal proteins on Ad genomes interferes with efficient DNA unwinding by the MRN complex, thereby making the virus genome a poor activator of ATM even though it is linear dsDNA. If this is the case, Ad DNA replication may be necessary to produce the ssDNA triggers needed for both ATM and ATR activation. Our results are consistent with the idea that incoming genomes may induce a limited set of DDR responses, including Mdc1 focus formation and the induction of focal concentrations of γH2AX (34), while widespread production of γH2AX (34) and the induction of Nbs1 and possibly also Chk1 phosphorylation may require viral DNA replication. In conclusion, it will be informative to dissect the ability of cellular DDR proteins to react to replication-defective and replication-competent Ad infections since this may provide new insights into how DNA damage is sensed and transduced to activate cellular responses.
ACKNOWLEDGMENTS
We are very grateful to Gary Ketner and Arnold Levine for providing antibodies used in this study. We thank Michel Perricaudet for providing AdRSVβgal. We also thank Gary Janssen for critically reading the manuscript and all of the members of our laboratory for their suggestions and support.
This research was supported by the National Cancer Institute (grant CA82111) and awards from Miami University.
FOOTNOTES
- Received 7 July 2012.
- Accepted 18 September 2012.
- Accepted manuscript posted online 26 September 2012.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.
