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Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection

Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection The EMBO Journal (2009) 28, 652–662 & 2009 European Molecular Biology Organization All Rights Reserved 0261-4189/09 | | T THE H E www.embojournal.org E E EMB M MB BO O O J JO OURN URN A AL L Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection 1,2,4,5 1,2,4 Christian T Carson , Nicole I Orazio , Introduction 1,4,6 1,7 Darwin V Lee , Junghae Suh , The cellular DNA damage sensing and repair machinery 3 1,5 Simon Bekker-Jensen , Felipe D Araujo , orchestrates cell-cycle checkpoints and DNA repair pathways 1,2 1 Seema S Lakdawala , Caroline E Lilley , (Kastan and Bartek, 2004; Harper and Elledge, 2007). At the 3 3 Jiri Bartek , Jiri Lukas and heart of the cellular response to DNA damage are the PI3 1, Matthew D Weitzman * kinase-like kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR), which phosphorylate multiple pro- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA, Graduate Program, Division of Biology, University tein targets. Although these kinases have many overlapping of California, San Diego, CA, USA and Centre for Genotoxic Stress substrates (Matsuoka et al, 2007; Stokes et al, 2007), they Research, Institute of Cancer Biology, Danish Cancer Society, respond differently to distinct types of damage. Recent Copenhagen, Denmark studies have begun to shed light on how these kinases are activated after DNA damage and replication stress (Lavin, The protein kinases ataxia-telangiectasia mutated (ATM) 2007; Lee and Paull, 2007; Zou, 2007; Burrows and Elledge, and ATM-Rad3 related (ATR) are activated in response to 2008). In response to DNA double-strand breaks (DSBs), ATM DNA damage, genotoxic stress and virus infections. Here is autophosphorylated on S1981 and subsequent dimer dis- we show that during infection with wild-type adenovirus, sociation yields active monomers (Bakkenist and Kastan, ATR and its cofactors RPA32, ATRIP and TopBP1 accumu- 2003). ATR responds to a broader spectrum of DNA damage late at viral replication centres, but there is minimal ATR substrates and is crucial for maintaining genomic stability activation. We show that the Mre11/Rad50/Nbs1 (MRN) during the cell-cycle S phase (Cimprich and Cortez, 2008). complex is recruited to viral centres only during infection ATR exists in a stable complex with an ATR-interacting with adenoviruses lacking the early region E4 and ATR protein ATRIP (Cortez et al, 2001) and is recruited to sites signaling is activated. This suggests a novel requirement of DNA damage by replication protein A (RPA) associated for the MRN complex in ATR activation during virus with single-stranded DNA (ssDNA) (Zou and Elledge, 2003). infection, which is independent of Mre11 nuclease activity It has been suggested that RPA-coated ssDNA is the substrate and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other that initiates ATR-mediated checkpoint signaling (Zou, 2007). damage scenarios, we found that ATM and ATR signaling TopBP1 is a mediator protein that binds and activates ATR/ are not dependent on each other during infection. We ATRIP complexes (Kumagai et al, 2006; Mordes et al, 2008). identify a region of the viral E4orf3 protein responsible Although ATM and ATR respond to different types of stimuli, for immobilization of the MRN complex and show that this they are integrated into a molecular circuit that links cellular prevents ATR signaling during adenovirus infection. We DNA replication machinery with DNA damage response propose that immobilization of the MRN damage sensor by pathways (Hurley and Bunz, 2007). E4orf3 protein prevents recognition of viral genomes and The Mre11, Rad50 and Nbs1 proteins form the MRN blocks detrimental aspects of checkpoint signaling during complex, which is involved in the cellular DNA damage virus infection. response (Stracker et al, 2004; Lavin, 2007). The MRN com- The EMBO Journal (2009) 28, 652–662. doi:10.1038/ plex is a sensor of DSBs (Petrini and Stracker, 2003) and is emboj.2009.15; Published online 5 February 2009 required for full activation of ATM in response to DSBs Subject Categories: genome stability & dynamics; microbiol- (Carson et al, 2003; Uziel et al, 2003; Horejsi et al, 2004; ogy & pathogens Lee and Paull, 2005, 2007). MRN binds directly to ATM and Keywords: adenovirus; ATR kinase; DNA damage response; stimulates kinase activity to phosphorylate substrates (Lee MRN complex and Paull, 2004; Falck et al, 2005; You et al, 2005). Although MRN complex function has been convincingly linked to ATM activation in response to DSBs, its role in ATR activation and downstream signaling in response to different types of da- mage is less clear. The MRN complex prevents DSBs during chromosomal replication (Costanzo et al, 2001) and is re- *Corresponding author. Laboratory of Genetics, The Salk Institute for quired for the intra S-phase checkpoint in response to repli- Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: þ 858 453 4100 Ext 2037; Fax: þ 858 558 7454; cation stress (Zhong et al, 2005; Olson et al, 2007b). DNA- E-mail: [email protected] damaging agents that stall replication induce hyperphosphor- These authors contributed equally to this work ylation of the middle subunit of RPA (RPA32) that modulates Present address: Becton Dickinson Biosciences, San Diego, CA, USA its activity (Binz et al, 2004; Liu et al, 2006). Nbs1 is required Present address: Pfizer, Groton, CT, USA Present address: Department of Bioengineering, Rice University, for ATR-induced hyperphosphorylation of RPA32 in response Houston, TX, USA to hydroxyurea (HU) (Manthey et al, 2007; Olson et al, 2007a). MRN has also been implicated in ATR activation Received: 18 August 2008; accepted: 23 December 2008; published and checkpoint signaling in response to UV treatment but online: 5 February 2009 652 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al may not be universally required for phosphorylation of all substrates at low doses (Difilippantonio et al, 2005; Zhong et al, 2005; Jazayeri et al, 2006; Myers and Cortez, 2006; Olson et al, 2007a). Many viruses interact with cellular DNA damage sensing and repair pathways (Lilley et al, 2007). In the case of human adenovirus (Ad), the cellular DNA repair machinery presents an obstacle to productive infection (Stracker et al, 2002). Infection with mutant Ad deleted of the E4 region results in formation of virus genome concatemers by a cellular DNA repair pathway (Boyer et al, 1999; Stracker et al, 2002). The MRN complex is required for concatemer formation, and wild-type Ad serotype 5 (Ad5) has evolved two ways to Figure 1 The Mre11 and Nbs1 proteins are required for robust ATR prevent concatemers by targeting the MRN complex signaling in response to mutant Ad infection. (A) Infections were (Stracker et al, 2002). The viral E1b55K and E4orf6 proteins performed in a transformed cell line deficient for Mre11 (A-TLD1) induce proteasome-mediated degradation of MRN proteins that was transduced with an empty retrovirus vector (vector) or a (Stracker et al, 2002; Carson et al, 2003), whereas expression vector expressing the wild-type Mre11 cDNA (Mre11). Cells were uninfected (Mock) or infected with wild-type Ad5 (Ad5) and the of the Ad5-E4orf3 protein mislocalizes MRN proteins into E4-deletion mutant (dl1004). Immunoblotting of lysates prepared at intranuclear track-like structures and cytoplasmic aggregates 30 hpi used antibodies for total protein (Chk1, RPA32, Mre11, Rad50 (Stracker et al, 2002, 2005; Araujo et al, 2005; Evans and and Nbs1) or phospho-specific sites (Chk1-S345). Ku86 served as a Hearing, 2005). loading control. (B) Infections were performed in the transformed NBS cell line (NBS-ILB1) transduced with an empty retrovirus Coincident with concatemer formation, infection with vector (vector) or a vector expressing the wild-type Nbs1 cDNA. E4-deleted Ad also elicits a cellular DNA damage response (Stracker et al, 2002; Carson et al, 2003). The ATM and ATR kinase signaling pathways are activated, as detected by autophosphorylation of ATM and phosphorylation of many versions of the A-TLD1 cell line that expresses mutant known ATM and ATR substrates (Carson et al, 2003). The Mre11 (Carson et al, 2003), and the NBS-ILB1 cell line that response is also characterized by recruitment of the MRN harbours an Nbs1 mutation (Cerosaletti et al, 2000). Lysates complex and other DNA damage response proteins to viral from cells infected with the E4-deleted virus dl1004 were replication centres. In contrast, this cellular DNA damage analyzed by immunoblotting with a phospho-specific anti- response is not observed during infection with wild-type Ad5 body to Chk1-S345, and with an antibody to RPA32 that expresses E4 proteins (Carson et al, 2003). Ad infection (Figure 1). Phosphorylation of Chk1 and RPA32 was detected provides a novel system to analyze the mechanism and in cell lines complemented with wild-type cDNAs. In con- consequences of ATR signaling. Here, we examine the effect trast, infection with E4-deleted Ad in the A-TLD1 (Figure 1A) of E4orf3-mediated MRN redistribution on ATM and ATR and NBS (Figure 1B) mutant lines produced significantly signaling. We show that activation of ATR signaling during reduced signaling. Wild-type Ad infection did not generate viral infection is dependent on the MRN complex but unlike these phosphorylation events due to degradation of MRN other types of damage is independent of ATM. The MRN proteins. These data show that the MRN complex is required complex is not required for accumulation of RPA, ATRIP, ATR for robust ATR signaling in response to Ad infection. and TopBP1 at viral replication centres. We examine MRN dynamics in living cells and show that E4orf3-induced im- The presence of Ad5-E4orf3 reduces ATR signaling but mobilization prevents MRN from responding to the virus and not ATM other types of DNA. These results show a novel role for MRN As the Ad5-E4orf3 protein affects MRN complex localization, in activation of ATR signaling independent of ATM and we investigated whether E4orf3 alters ATM and ATR signaling downstream of recruitment of RPA/ATR/ATRIP/TopBP1. during infection. Because the viral E1b55K and E4orf6 pro- teins downregulate damage signaling through degradation of the MRN complex (Carson et al, 2003), we tested a mutant Results that is deleted of E4orf6 and E1b55K but retains E4orf3 The MRN complex is required for robust ATR signaling (dl1017) (Bridge and Ketner, 1990), and compared it with during infection wild-type Ad5 and the large E4-deleted mutant (see We have showed earlier the phosphorylation of ATM and ATR Figure 2A). The E4-deleted virus induced phosphorylation kinase substrates in response to E4-deleted Ad infection of ATR substrates Chk1 and RPA32, as detected by a (Carson et al, 2003). Some sites were modified by either shift in the mobility of RPA32 and by recognition with ATM or ATR (e.g., Chk2-T68 and Nbs1-S343), whereas others phospho-specific antibodies (Chk1-S345 and RPA32-S4,8) were ATR specific (e.g., Chk1-S345 and RPA32). We showed (Figure 2B). These phosphorylation events were greatly that MRN is required for ATM activation (Carson et al, 2003), reduced during infection with dl1017 that expresses E4orf3, but the requirement for MRN in ATR signaling during virus indicating that ATR signaling is reduced in the presence of infection has not been clarified. To investigate the role of Ad5-E4orf3. MRN specifically in ATR-dependent signaling responses to We also examined the effect of E4orf3 on ATM signaling. In virus, we examined phosphorylation events during infections contrast to ATR, ATM signaling was intact during infection of cells with hypomorphic mutations in Mre11 and Nbs1. with dl1017, as evidenced by ATM autophosphorylation on Infections were performed in mutant and complemented S1981 (Figure 2B). Analysis of substrates that are phosphory- &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 653 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 2 Phosphorylation of ATR substrates is reduced in the presence of Ad5-E4orf3 and is independent of ATM. (A) Genotypes of mutant viruses used for infection (Bridge and Ketner, 1990). (B) HeLa cells were mock infected or infected with indicated viruses. Cells were harvested at 6, 12, 18 and 24 hpi and prepared for analysis by immunoblotting using antibodies to phospho-specific residues (Chk1-S345, RPA32-S4,8 and ATM-S1981). Below are lysates at 24 hpi probed with antibodies against total protein (Chk1, RPA32 and Ku86) to serve as loading controls. (C) Immunoblots of infections in control cells (48BR) or Seckel cells with mutant ATR (GM18366). (D) Immunoblots of infections in A-T cells with mutant ATM (AT221JE-T) or a complemented line (A-T þATM). DBP was a marker of virus infection and Ku86 served as a loading control. lated by both ATM and ATR (Chk2-T68, Nbs1-S434 and Accumulation of ATR at viral replication centres is BRCA1) revealed that their phosphorylation was decreased independent of MRN but not abolished by E4orf3 expression (Supplementary Viral replication centres can be detected by staining with Figure S1A), showing that E4orf3 inhibits ATR but not antibodies to the viral DNA-binding protein (DBP) that coats ATM. Immunofluorescence during infection with dl1017 ssDNA accumulated at viral replication sites (Pombo et al, showed that MRN mislocalization by E4orf3 did not prevent 1994). The cellular RPA32 protein also accumulates at these ATM activation, and that phosphorylated Nbs1 colocalized sites (Stracker et al, 2005), with both wild-type and mutant with Rad50 in nuclear tracks (Supplementary Figure S1B). viruses (Figure 3A). The E4orf3 protein is excluded from viral Together, these data suggest that MRN mislocalization by replication centres and appears in intranuclear tracks and E4orf3 is not sufficient to prevent ATM activation, indicating cytoplasmic aggregates (Araujo et al, 2005; Evans and that ATM and ATR signaling pathways can be independently Hearing, 2005), with both wild-type Ad5 or the dl1017 mutant regulated during Ad infection. (Figure 3A). The Ad5-E4orf3 protein is sufficient to redistri- To determine the interdependence of ATR and ATM, we bute the MRN complex, as detected by staining for Nbs1 examined signaling in cells with kinase mutations. Cells (Figure 3A). In contrast, infection with dl1004 leads to derived from a Seckel syndrome patient with mutation in accumulation of MRN at viral centres. We, therefore, inves- ATR (O’Driscoll et al, 2003) and from an A-T patient with tigated whether MRN mislocalization by E4orf3 correlates mutation in ATM were infected with viruses that express with inhibition of ATR signaling (Figure 3B). Phosphorylation E4orf3 (Ad5 or dl1017) and those that lack E4orf3 (dl1004 or of RPA32 at viral centres, as detected by staining with the dl1016). Phosphorylation of Chk1-S345 and RPA32-S4,8 was RPA32-S4,8 antibody, was only observed when the MRN abolished in the Seckel cells (Figure 2B). ATM activation complex was associated with viral replication centres (i.e., and signaling were intact in Seckel cells, as detected by in the absence of E4orf3 during infection with dl1004). When autophosphorylation on S1981 (Figure 2C) and phosphory- the virus expressed E4orf3, RPA32 phosphorylation was not lation of downstream substrates (Supplementary Figure S1C). detected. This shows that E4orf3 mislocalization of the MRN Infections of A-T cells or a matched complemented line with complex prevents its accumulation at viral replication cen- the mutant virus dl1016 that lacks both E1b55K and E4orfs tres, and that this correlates with the absence of ATR signal- 1-3 (DE1b55K/DE4orf1-3) induced ATR signaling (Figure 2C). ing to RPA32. However, signaling to Chk1 and RPA32 was abrogated in the We also investigated whether E4orf3 affects localization of presence of E4orf3 (dl1017). These results show that ATR other proteins involved in ATR signaling. We observed that activation in response to Ad infection, and its inhibition by ATR, RPA and ATRIP accumulated at viral replication centres E4orf3, are independent of ATM. with all viruses tested, irrespective of E4orf3 expression 654 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 3 Signaling by ATR at viral replication centres. (A) E4orf3 sequesters Nbs1 into tracks away from viral replication centres. HeLa cells were mock infected or infected with indicated viruses. Cells were fixed at 16 hpi, and immunofluorescence was performed. Staining shows that Nbs1 but not RPA32 is excluded from viral replication centres in the presence of E4orf3. (B) Colocalization of the MRN complex with RPA32 at replication centres correlates with RPA32 phosphorylation. Immunofluorescence with a phospho-specific antibody to RPA32-S4,8 shows colocalization with viral replication centres stained with an antibody to DBP only in the absence of E4orf3. (C) Accumulation of RPA32, ATR and ATRIP at viral centres is independent of E4. (D) TopBP1 accumulates at viral centres independently of E4. (E) Activation is observed for ATM, but not ATR, in Seckel cells. (F) ATR signaling is restored in A-TLD cells by expression of Mre11 or the nuclease-defective mutant Mre11-3. In all images, the nuclei were located by co-staining cellular DNA with DAPI (blue). (Figure 3C) (Carson et al, 2003; data not shown). These data activity through an interaction with ATRIP (Kumagai et al, suggest that E4orf3 does not abrogate ATR signaling by 2006; Mordes et al, 2008). We examined localization of mislocalizing ATR, RPA32 or ATRIP. It also shows that the TopBP1 during infection and found it localized in viral DBP presence of ATR at viral centres is not sufficient to initiate or centres with wild-type and E4 mutant viruses (Figure 3D). maintain the DNA damage signaling during infection. The Immunofluorescence of infected Seckel cells confirmed that TopBP1 protein is a regulator of ATR that stimulates kinase phosphorylation of RPA32-S4,8 was not observed in the &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 655 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al absence of functional ATR, although RPA32 and ATRIP basis of these alignments, we converted isoleucine at position accumulated at viral centres (Figure 3E). Lack of RPA32 104 of Ad5 to arginine, which is present in all other serotypes phosphorylation was confirmed in Seckel cells from multiple (Figure 4A). The Ad5 E4orf3-I104R mutant retained the origins and could also be restored by introduction of the wild- ability to form nuclear tracks and disrupt PML but was type ATR gene (Supplementary Figure S2). ATM activation defective for redistribution of the MRN complex was detectable in Seckel cells as revealed by staining for (Figure 4B). This identified a region of E4orf3 involved in ATM-S1981 and is therefore independent of ATR in response targeting the MRN complex and provided a separation-of- to Ad infection (Figure 3E). Although RPA and ATR were function mutant. detected at viral centres in the absence of functional MRN We next examined the ability of Ad5-E4orf3, Ad12-E4orf3 complex, phosphorylation of RPA32 was not observed and E4orf3-I104R proteins to prevent ATR signaling during during infections of NBS and A-TLD1 cells (Figure 3F, infection with E4-deleted virus (Figure 5). The cells were Supplementary Figure S2C and D). ATR signaling was re- transfected with E4orf3 expression vectors, and then infected stored when infections were performed in A-TLD1 cells with dl1004. Immunoblotting revealed abrogated ATR signal- transduced with retroviruses that express either wild-type ing in the presence of Ad5-E4orf3 (reduced Chk1-S345 and Mre11 or the nuclease-defective Mre11-3 mutant (Figure 3F). RPA32-S4,8 phosphorylation and RPA32 mobility shift), Together, these data show that the MRN complex is not although ATM autophosphorylation was not significantly required for localization of ATR with ssDNA at viral replica- altered (Figure 5A). In contrast, the Ad12-E4orf3 and tion centres but is required for ATR signaling. E4orf3-I104R proteins, which failed to mislocalize the MRN complex, did not inhibit ATR signaling (Figure 5A and B). In Mislocalization of the MRN complex by Ad5-E4orf3 is this experiment, there was a slight decrease in ATM signaling required for disruption of ATR signaling (Figure 5B), but this was not reproducible and did not Although E4orf3 proteins from all serotypes tested form correlate with MRN mislocalization. Cells from the same nuclear tracks, rearrange the promyelocytic leukemia protein experiment were also examined by immunofluorescence for RPA32-S4,8 phosphorylation, as a marker of ATR signaling PML and redistribute components of the PML bodies, only Ad5-E4orf3 mislocalizes the MRN complex (Stracker et al, (Figure 5C). We observed that Ad5-E4orf3, but not Ad12- 2005). Sequence analysis for the highly conserved E4orf3 E4orf3 or E4orf3-I104R, prevented RPA phosphorylation at proteins revealed residues specific to the subgroup C viruses viral replication centres. Cotransfection of a plasmid expres- (Ad1, Ad2 and Ad5) that uniquely mislocalize MRN. On the sing GFP served as a marker for transfected cells. Control Figure 4 A region in Ad5-E4orf3 important for targeting the MRN complex. (A) Sequence alignment of E4orf3 proteins. E4orf3 sequences from different human (subgroup noted in brackets) and simian adenoviruses were aligned using the CLUSTAL algorithm and the region around residue I104 is shown. Conserved residues are shown boxed, with CLUSTAL colour scheme reflecting amino acids of similar chemical nature. The I104 residue is highlighted, showing that this site differs between subgroup C and all other sequenced E4orf3 genes. (B) The Ad5 E4orf3- I104R mutant does not redistribute the MRN complex. Plasmids for wild-type and mutant E4orf3 were transfected into HeLa cells. Immunofluorescence shows that the E4orf3 mutant protein still forms tracks and disrupts PML structures but is unable to redistribute members of the MRN complex, which remain diffusely nuclear. Representative images are shown and nuclei are located by co-staining cellular DNA with DAPI. 656 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al structures and accumulated in cytoplasmic aggregates (Figure 6A). Both Mre11–YFP and Nbs1–YFP fusion protein colocalized with endogenous Rad50 protein. Similar results were obtained using a stable cell line expressing Nbs1–2GFP (Lukas et al, 2003) (data not shown). These results show that the fusion proteins are correctly incorporated into E4orf3- induced structures. During infection with E4-deleted virus, Nbs1–YFP formed foci at viral replication centres (Figure 6A), as reported earlier for endogenous MRN (Stracker et al, 2002). We then examined the dynamics of MRN in E4orf3 tracks using fluorescence recovery after photobleaching (FRAP) to determine the rate at which a bleached area is repopulated with new fluorescent protein. As reported earlier, we ob- served that the Mre11 and Nbs1 proteins are highly mobile in the absence of E4orf3 (Figure 6B). FRAP analysis of Nbs1 foci at viral replication centres during infection with E4-deleted virus dl1004 showed that the signal recovered with kinetics slower than that of an undamaged area (Figure 6C). The increased residence time at virus centres resembles that observed for recruitment of the MRN complex at DSBs (Lukas et al, 2003). In contrast, the kinetics of fluorescence recovery was much more significantly affected by the pre- sence of E4orf3, where the fluorescence signal did not recover after photo-bleaching of MRN in tracks induced by E4orf3 during transfection (Figure 6B) or infection (Figure 6C). We also compared MRN mobility in the presence of wild-type and I104R mutant E4orf3 proteins (Figure 6D). Although wild-type Ad5-E4orf3 expression led to immobilization of Mre11–YFP, the I104R mutant barely affected Mre11 dy- namics. This supports our observations from fixed images that show I104R does not alter Mre11 localization. Together, Figure 5 ATR signaling in response to virus infection is abrogated these data show that MRN in tracks induced by Ad5-E4orf3 by E4orf3 proteins that mislocalize the MRN complex. HeLa cells represent immobilized proteins. were transfected with an empty plasmid (vector) or with vectors expressing E4orf3 proteins. After 24 h, cells were either mock E4orf3 prevents ATR-dependent signaling in response infected, infected with the E1b55K/E4orf6 mutant (dl1017) (positive control), or infected with the E4-deletion mutant (dl1004). (A, B) to nonviral damage Lysates from cells at 24 hpi were immunoblotted with antibodies to To assess the impact of MRN immobilization on the ability of Chk1-S345, RPA32-S4,8, ATM-1981 and RPA32. (C) Cells were fixed cells to respond to DNA damage, we generated spatially at 18 hpi, and immunofluorescence was performed with an anti- restricted DSBs through laser microirradiation (Lukas et al, body to RPA32-S4,8. Images are shown merged with DAPI staining. A plasmid expressing GFP was cotransfected with that for E4orf3 at 2003). Local DNA damage was generated in subnuclear a ratio of 1:10 and GFP staining is shown in the lower left insert regions by a focussed laser beam programmed to move panel as a positive control for transfection. once across individual cell nuclei. Previous studies using visualization with specific antibodies and fluorescently tagged proteins have shown that DNA damage proteins are staining showed that viral DBP centres were formed in all redistributed to these DSBs (Lukas et al, 2003, 2004). We cells, and that expression of E4orf3 proteins resulted in PML examined recruitment of endogenous cellular repair proteins disruption (data not shown). Together, these data support the to laser-induced DSBs in cells expressing E4orf3. The sites of hypothesis that mislocalization of the MRN complex by microirradiation can be visualized by recruitment of the E4orf3 leads to abrogation of ATR signaling. mediator protein Mdc1 (Lukas et al, 2004), which is unaf- fected by E4orf3 (Figure 7A). Cells expressing Ad5-E4orf3 The MRN complex is immobilized by Ad5-E4orf3 localized endogenous Nbs1 into the characteristic nuclear We reported earlier that Ad5-E4orf3 expression alters MRN tracks and displayed minimal accumulation of Nbs1 at micro- complex solubility (Araujo et al, 2005). To examine the effect irradiation sites compared with neighbouring untransfected of E4orf3 on protein dynamics in live cells, we used fluores- cells. This result shows that Nbs1 immobilization in E4orf3- cently tagged fusion proteins of Mre11 and Nbs1. We ex- induced tracks prevents its accumulation at DSBs. pressed Mre11–YFP and Nbs1–YFP by cell transfection either To examine the effect of E4orf3 expression on kinetics of in the presence of E4orf3 alone or in virus-infected cells the DNA damage response, we combined laser microirradia- (Figure 6). The fusion proteins were distributed diffusely in tion with live cell imaging (Figure 7B). The Nbs1–2GFP cell the nucleoplasm in untreated cells (Lukas et al, 2003). In the line was cotransfected with the Ad5-E4orf3 expression vector presence of E4orf3, provided by virus infection or plasmid and a plasmid expressing RFP to serve as a transfection transfection, the fusion proteins localized into track-like marker. Generation of subnuclear restricted DSBs resulted &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 657 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 6 E4orf3 abrogates MRN function by immobilizing the MRN complex. (A) HeLa cells were transfected with Nbs1–YFP or Mre11–YFP alone or together with Ad5-E4orf3 plasmid vector. After 24 h, cells were infected with dl1004 or dl1017. Cells were fixed 18 hpi, and immunofluorescence was performed with a Rad50 antibody. Images shown are merged with DAPI staining. (B–D) FRAP analysis of Nbs1 and Mre11 in cells expressing E4orf3 by infection or transfection. The unbleached portion of the cell served to normalize the overall fluorescence decay during the repeated image collection. Arrows above indicate the time of bleaching. (B) Stable U2OS cells expressing Nbs1–2GFP were either untreated, transfected with E4orf3, or treated with 10 Gy gamma-irradiation. (C) HeLa cells were transfected with Nbs1–YFP and then mock treated or infected with dl1004 and dl1017. Cells were analyzed by FRAP at 18 hpi. (D) HeLa cells transfected with Mre11–YFP together with either empty vector or vectors expressing Ad5-E4orf3 and E4orf3-I104R were analyzed by FRAP. in rapid recruitment of Nbs1–2GFP. In contrast, there was report, we show that MRN also has an important function barely detectable recruitment of Nbs1 in cells expressing in ATR signaling in response to E4-deleted Ad. Substrates Ad5-E4orf3, even at 15 min after treatment. These data known to contribute to ATR kinase activation include ssDNA indicate that the MRN complex immobilized in E4orf3- coated with RPA, and junctions of single- and double- induced tracks is unable to respond efficiently to exogenous stranded DNA (MacDougall et al, 2007; Zou, 2007). Our DNA damage. As MRN has been shown to be required for data show that ssDNA at viral replication centres (Pombo ATR signaling in response to HU treatment (Manthey et al, et al, 1994) is sufficient for recruitment of ATR and ATRIP, 2007), we used recombinant Ad vectors to express but that ATR signaling to Chk1 and RPA32 is only detected Ad5-E4orf3 or GFP in cells and then exposed them to HU when the MRN complex also accumulated at viral centres. (Figure 7C). Signaling was abrogated by E4orf3 as shown by Although we have focussed on MRN, we cannot exclude decreased hyperphosphorylation of RPA32 and staining additional roles for other proteins implicated in ATR activa- with phospho-specific antibodies to RPA32-S4,8, Nbs1-S343 tion and checkpoint signaling, such as the 9-1-1 complex, the (Figure 7C), and Chk1-S345 (data not shown). Together, these Rad17 complex and Claspin (Zou, 2007). The cellular data show that immobilization of MRN by E4orf3 prevents E1b55K-associated protein E1B-AP5 was recently implicated the ATR-mediated response to replication stress. in ATR signaling during Ad infection (Blackford et al, 2008), but this factor localizes to wild-type Ad5 centres and, there- fore, does not explain the induction of ATR signaling in the Discussion absence of E4. The role of MRN in ATR activation MRN has recently been implicated in facilitating ATR The MRN complex has an important function in the cellular activation and signaling in response to some types of damage. DNA repair response to Ad infection. We showed earlier that Processing of DSBs in an MRN-dependent manner results in the formation of ssDNA and ATR activation (Adams et al, during infection with E4-deleted Ad, MRN is required for 2006; Jazayeri et al, 2006; Myers and Cortez, 2006). MRN concatemerization of the viral genome and activation of ATM is involved in ATR-mediated phosphorylation events in signaling (Stracker et al, 2002; Carson et al, 2003). In this 658 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 7 E4orf3 prevents ATR-dependent damage signaling induced by nonviral sources. (A) U2OS cells transfected with a plasmid vector expressing Ad5-E4orf3 were laser microirradiated. Cells were fixed and stained for endogenous Nbs1 and Mdc1. Arrows indicate cells with E4orf3-induced tracks of Nbs1. (B) Stable U2OS cell lines expressing Nbs1–2GFP were transfected with a plasmid expressing Ad5-E4orf3, together with prRFP-C1, which was used as a marker for transfected cells. Cells were laser microirradiated (as indicated by dashed line) and images show the recruitment of Nbs1 to sites of damage. (C) HeLa cells were mock infected, or infected with E1-deleted recombinant Ads expressing GFP (rAd-GFP) or E4orf3 (rAd-E4orf3). At 24 hpi, the cells were mock treated or treated with 2 mM hydroxyurea (HU) for 2 h, and the cells were harvested for immunoblotting. Cellular proteins were detected with antibodies to RPA32 and specific phosphorylated sites at RPA-S4,8 and Nbs1-S343. Ku86 served as a loading control. response to replication stress, although signaling events may point (Robison et al, 2004; Olson et al, 2007b) and also also be MRN independent, depending on the substrate and interacts with ATR/ATRIP (Olson et al, 2007a). Either of dose of damaging agent (Pichierri and Rosselli, 2004; Stiff these two interactions could contribute to ATR activation by et al, 2005; Zhong et al, 2005; Olson et al, 2007b). During MRN at viral centres. The ATM and ATR kinases may be infection with E4-deleted Ad, it is not clear which DNA coordinated and interdependent in response to some types of structures serve as the trigger for ATR signaling, and there damage (Hurley and Bunz, 2007). In response to IR, activa- may be multiple ways that the MRN complex contributes to tion of ATR is ATM dependent (Jazayeri et al, 2006; Myers ATR activation. Although the nuclease activity of Mre11 is and Cortez, 2006), whereas in response to HU and UV required for joining Ad genomes into concatemers (Stracker activation of ATM is ATR dependent (Liu et al, 2005; Stiff et al, 2002), we found that it was not required for ATR et al, 2006). In the case of virus infection, we have found that signaling. In contrast to DSBs caused by IR or replication although both rely upon the MRN complex, ATM and ATR stress, where the nuclease activity of Mre11 is required for signaling are independent of each other. This may reflect the resection (Buis et al, 2008), we found that Mre11 nuclease fact that during infection there are numerous substrates for activity is not required for generation of ssDNA and recruit- kinase activation, including replication intermediates and ment of RPA/ATR/ATRIP at viral centres. However, signaling double-strand ends. In addition to the viral genome, infection may be triggered by further processing of the viral genome, may also induce chromosomal damage to the host genome. for example, by removal of the terminal protein from the 5 Early Ad genes alter cell-cycle progression, which could lead end of the genome by other nucleases to generate free DNA to collapse of replication forks, and also cause genomic ends. MRN-dependent processing of DSBs has been sug- instability and chromosomal aberrations (Caporossi and gested to generate small oligonucleotides that stimulate Bacchetti, 1990; Lavia et al, 2003). Therefore, although the ATM activity (Jazayeri et al, 2008). It will be interesting to phosphorylated ATR substrates predominantly accumulate at determine whether these are generated during virus infection viral replication centres, it is possible that chromosomal and whether they play a role in ATR activation. The MRN damage also contributes to induction of ATR signaling during complex could have a direct role in stimulating ATR kinase infection. activity, as has been shown in vitro for ATM (Lee and Paull, 2004). The MRN complex may also facilitate phosphorylation The function of viral E4 proteins of downstream substrates through recruitment or retention of There is functional redundancy between the E4orf3 and proteins to viral centres, as has been proposed for stalled E4orf6 products from Ad, and either is sufficient to promote replication forks (Stiff et al, 2005). MRN associates with RPA viral replication, prevent concatemerization of the viral at sites of DNA damage to mediate the intra-S-phase check- genome, and enable viral late protein production. Both E4 &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 659 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al proteins target the MRN complex to prevent concatemer (Wilkinson and Weller, 2006). Induction of the Epstein–Barr formation (Stracker et al, 2002; Evans and Hearing, 2003). virus lytic program also elicits a cellular DNA damage re- Together with previous observations of MRN degradation by sponse and ATM activation, but ATR signaling is minimal E1b55K/E4orf6 (Carson et al, 2003), our data show that both (Kudoh et al, 2005). Therefore, viruses appear to use multiple E4 proteins target MRN to prevent damage signaling. strategies to inactivate ATR and downstream signaling events Inactivation of MRN is also likely to be responsible for the during viral infection, to prevent negative impacts of the ability of E4 proteins to promote viral DNA replication. We cellular response to replication stress on virus production. and others have found that MRN inhibits replication of E4- Understanding how viruses such as Ad manipulate signaling deleted mutant Ad, although the mechanism is unclear pathways will provide insights into the regulation of DNA (Evans and Hearing, 2005; Mathew and Bridge, 2007, 2008; damage responses in mammalian cells. Lakdawala et al, 2008). Replication of cellular DNA is tightly regulated to ensure that the genome is replicated only once Materials and methods per cell cycle (Arias and Walter, 2007). MRN is recruited to cellular replication origins and can inhibit firing of new Cell lines origins of DNA replication upon damage (Olson et al, HeLa and 293 cells were purchased from the American Tissue 2007b) and suppress rereplication (Wu et al, 2004; Lee Culture Collection. W162 cells for growth of E4-deleted viruses were from G Ketner, A-T cells (AT221JET and complemented et al, 2007). Mre11 has recently been suggested to bind the version) were from Y Shiloh, and Seckel cells were from Coriell Ad genome (Mathew and Bridge, 2008), but it is unclear how Institute (GM18366) and A D’Andrea (F02-98). Immortalized this inhibits replication. Checkpoint signaling by ATM and A-TLD1 and NBS (NBS-ILB1) and matched cells reconstituted with ATR is not responsible for the defective replication of E4- wild-type Mre11 and NBS1 were described (Cerosaletti et al, 2000; Carson et al, 2003). The retrovirus expression plasmid for the deleted Ad (Lakdawala et al, 2008). The virus uses its own Mre11-3 mutant (HD129/130LV) was generated by site-directed protein-priming mechanism and polymerase, which could be mutagenesis and A-TLD1 cells were transduced by the retrovirus as affected by MRN binding to the origin or its participation in described previously (Carson et al, 2003). The U2OS-derived stable removal of the terminal protein from the viral genome. cell line with Nbs1–2GFP has been described (Lukas et al, 2003). Cells were maintained as monolayers in either Dulbecco modified Our work links the E4orf3-induced redistribution of pro- Eagle’s medium (DMEM) or MEM plus Earle’s salts (Seckel cells) teins associated with PML nuclear bodies to their role in supplemented with 10 or 20% fetal bovine serum (FBS), at 371Cin a sensing DNA damage (Everett, 2006). We show that reloca- humidified atmosphere containing 5% CO . lization of the MRN complex dramatically reduces its dynamics, essentially immobilizing the proteins in E4orf3- Plasmids and transfections induced intranuclear tracks. Similar observations were made Expression vectors for Ad5-E4orf3 and Ad12-E4orf3 proteins were with FRAP analysis of other fluorescently tagged components described (Stracker et al, 2005). Site-directed mutagenesis of E4orf3 was performed using QuikChange (Stratagene). Cells were trans- of the PML bodies (unpublished observations). Experiments fected with Lipofectamine 2000 (Invitrogen) according to manu- with laser microirradiation and live cell imaging showed that facturer’s protocol. recruitment of MRN to damage sites was severely abrogated in cells expressing Ad5 E4orf3. This correlated with decreased Viruses and infections recruitment of RPA and ATR, as well as abrogated damage The mutant viruses dl1004 (DE4), dl1016 (DE4orf1-3/DE1b55K) and signaling, and was not seen with the I104R mutant (data not dl1017 (DE4orf6/DE1b55K) have been described (Bridge and Ketner, 1990) and were obtained from G. Ketner. Wild-type Ad5 and dl1017 shown). Together, these results show that immobilization by were propagated in 293 cells. The dl1004 and dl1016 viruses were E4orf3 prevents the MRN damage sensor from responding to propagated on W162 cells (Weinberg and Ketner, 1983). All viruses new damage sites. This supports the hypothesis that seques- were purified by two sequential rounds of ultra-centrifugation in tering MRN (and other host factors) into E4orf3-induced cesium chloride gradients and stored in 40% glycerol at 201C. Infections were performed in DMEM supplemented with 2% FBS. tracks will prevent these proteins from sensing and accumu- After 2 h at 371C additional serum was added to a total of 10%. lating at virus centres and will thus thwart host antiviral responses. It has also been suggested that sequestration of Antibodies MRN in cytoplasmic aggresomes by the adenoviral E1b55K Primary antibodies were purchased from Novus Biologicals Inc. inactivates the complex and protects the viral genome (Liu (Nbs1), Genetex (Mre11-12D7, Rad50-13B3), Cell Signaling (Chk1- et al, 2005). As the E4orf3 and E4orf6 proteins both block S345), Rockland (ATM S1981-P), Santa Cruz (ATR, PML, Chk1, Ku86), Upstate Biotechnology (ATRIP), BD Bioscience (TopBP1) damage signaling and also promote production of late viral and Bethyl (RPA32-S4,8). The antibody to RPA32 was from proteins, it will be interesting to determine whether these T. Melendy, the monoclonal B6 antibody to DBP was from activities are linked. A. Levine, polyclonal rabbit antisera to DBP was from P. van der Inactivation of ATR may be a general approach used by Vliet, and the E4orf3 antibody was from T. Dobner. Secondary antibodies were from Jackson Laboratories, Eurogentec and viruses to neutralize aspects of the host defense. In addition Invitrogen Molecular Probes. to our observations with Ad infection, it has been suggested that ATR signaling is manipulated by other DNA viruses. For Immunoblotting and immunofluorescence example, in the case of herpes-simplex virus type 1 (HSV-1), Immunoblotting and immunofluorescence were performed as infection activates ATM signaling pathways and results in described previously (Carson et al, 2003). Novex (Invitrogen) 3 to accumulation of cellular repair proteins at viral centres (Lilley 8% gradient gels were used for the resolution of ATM. For et al, 2007). Although RPA is found at HSV-1 viral replication immunofluorescence, cells grown on glass coverslips were infected at an MOI of 25–100 pfu/cell. After 16–24 h, the cells were washed, compartments, ATR-dependent signaling is not activated, and fixed, stained and counter-stained with 4 ,6-diamidino-2-phenylin- it has been suggested that it is prevented because of spatial dol (DAPI). Immunoreactivity was visualized using a Nikon uncoupling of the ATR-ATRIP complex and sequestering of microscope in conjunction with a CCD camera (Cooke Sensicam) phosphorylated RPA in viral-induced nuclear domains or a Leica confocal microscope. 660 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al FRAP analysis reagents. We thank members of the Weitzman lab, past and present, FRAP analysis was performed in the Nbs1–2GFP U2OS cells as for discussions and critical reading of the manuscript. We thank D reported previously (Lukas et al, 2003). Further details are provided Ornelles for discussions and sequence analysis for E4orf3. We in Supplementary data. Image collection and FRAP data were acknowledge the James B Pendleton Charitable Trust for providing processed on a Leica confocal microscope. the Pendleton Microscopy Facility. This work was supported by NIH grant CA97093 (MDW), and by gifts from the Joe W & Dorothy Supplementary data Dorsett Brown Foundation and the Lebensfeld Foundation to MDW. Supplementary data are available at The EMBO Journal Online Additional support came from The Danish National Research (http://www.embojournal.org). Foundation, Danish Cancer Society, European Commission (DNA Repair) and the John and Birthe Meyer Foundation. CTC was supported by the Timken-Sturgis Foundation and a scholarship Acknowledgements from the ARCS Foundation. CTC and NIO were supported in part by an NIH Training Grant to the Salk Institute. 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Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection

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Abstract

The EMBO Journal (2009) 28, 652–662 & 2009 European Molecular Biology Organization All Rights Reserved 0261-4189/09 | | T THE H E www.embojournal.org E E EMB M MB BO O O J JO OURN URN A AL L Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection 1,2,4,5 1,2,4 Christian T Carson , Nicole I Orazio , Introduction 1,4,6 1,7 Darwin V Lee , Junghae Suh , The cellular DNA damage sensing and repair machinery 3 1,5 Simon Bekker-Jensen , Felipe D Araujo , orchestrates cell-cycle checkpoints and DNA repair pathways 1,2 1 Seema S Lakdawala , Caroline E Lilley , (Kastan and Bartek, 2004; Harper and Elledge, 2007). At the 3 3 Jiri Bartek , Jiri Lukas and heart of the cellular response to DNA damage are the PI3 1, Matthew D Weitzman * kinase-like kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR), which phosphorylate multiple pro- Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA, Graduate Program, Division of Biology, University tein targets. Although these kinases have many overlapping of California, San Diego, CA, USA and Centre for Genotoxic Stress substrates (Matsuoka et al, 2007; Stokes et al, 2007), they Research, Institute of Cancer Biology, Danish Cancer Society, respond differently to distinct types of damage. Recent Copenhagen, Denmark studies have begun to shed light on how these kinases are activated after DNA damage and replication stress (Lavin, The protein kinases ataxia-telangiectasia mutated (ATM) 2007; Lee and Paull, 2007; Zou, 2007; Burrows and Elledge, and ATM-Rad3 related (ATR) are activated in response to 2008). In response to DNA double-strand breaks (DSBs), ATM DNA damage, genotoxic stress and virus infections. Here is autophosphorylated on S1981 and subsequent dimer dis- we show that during infection with wild-type adenovirus, sociation yields active monomers (Bakkenist and Kastan, ATR and its cofactors RPA32, ATRIP and TopBP1 accumu- 2003). ATR responds to a broader spectrum of DNA damage late at viral replication centres, but there is minimal ATR substrates and is crucial for maintaining genomic stability activation. We show that the Mre11/Rad50/Nbs1 (MRN) during the cell-cycle S phase (Cimprich and Cortez, 2008). complex is recruited to viral centres only during infection ATR exists in a stable complex with an ATR-interacting with adenoviruses lacking the early region E4 and ATR protein ATRIP (Cortez et al, 2001) and is recruited to sites signaling is activated. This suggests a novel requirement of DNA damage by replication protein A (RPA) associated for the MRN complex in ATR activation during virus with single-stranded DNA (ssDNA) (Zou and Elledge, 2003). infection, which is independent of Mre11 nuclease activity It has been suggested that RPA-coated ssDNA is the substrate and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other that initiates ATR-mediated checkpoint signaling (Zou, 2007). damage scenarios, we found that ATM and ATR signaling TopBP1 is a mediator protein that binds and activates ATR/ are not dependent on each other during infection. We ATRIP complexes (Kumagai et al, 2006; Mordes et al, 2008). identify a region of the viral E4orf3 protein responsible Although ATM and ATR respond to different types of stimuli, for immobilization of the MRN complex and show that this they are integrated into a molecular circuit that links cellular prevents ATR signaling during adenovirus infection. We DNA replication machinery with DNA damage response propose that immobilization of the MRN damage sensor by pathways (Hurley and Bunz, 2007). E4orf3 protein prevents recognition of viral genomes and The Mre11, Rad50 and Nbs1 proteins form the MRN blocks detrimental aspects of checkpoint signaling during complex, which is involved in the cellular DNA damage virus infection. response (Stracker et al, 2004; Lavin, 2007). The MRN com- The EMBO Journal (2009) 28, 652–662. doi:10.1038/ plex is a sensor of DSBs (Petrini and Stracker, 2003) and is emboj.2009.15; Published online 5 February 2009 required for full activation of ATM in response to DSBs Subject Categories: genome stability & dynamics; microbiol- (Carson et al, 2003; Uziel et al, 2003; Horejsi et al, 2004; ogy & pathogens Lee and Paull, 2005, 2007). MRN binds directly to ATM and Keywords: adenovirus; ATR kinase; DNA damage response; stimulates kinase activity to phosphorylate substrates (Lee MRN complex and Paull, 2004; Falck et al, 2005; You et al, 2005). Although MRN complex function has been convincingly linked to ATM activation in response to DSBs, its role in ATR activation and downstream signaling in response to different types of da- mage is less clear. The MRN complex prevents DSBs during chromosomal replication (Costanzo et al, 2001) and is re- *Corresponding author. Laboratory of Genetics, The Salk Institute for quired for the intra S-phase checkpoint in response to repli- Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: þ 858 453 4100 Ext 2037; Fax: þ 858 558 7454; cation stress (Zhong et al, 2005; Olson et al, 2007b). DNA- E-mail: [email protected] damaging agents that stall replication induce hyperphosphor- These authors contributed equally to this work ylation of the middle subunit of RPA (RPA32) that modulates Present address: Becton Dickinson Biosciences, San Diego, CA, USA its activity (Binz et al, 2004; Liu et al, 2006). Nbs1 is required Present address: Pfizer, Groton, CT, USA Present address: Department of Bioengineering, Rice University, for ATR-induced hyperphosphorylation of RPA32 in response Houston, TX, USA to hydroxyurea (HU) (Manthey et al, 2007; Olson et al, 2007a). MRN has also been implicated in ATR activation Received: 18 August 2008; accepted: 23 December 2008; published and checkpoint signaling in response to UV treatment but online: 5 February 2009 652 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al may not be universally required for phosphorylation of all substrates at low doses (Difilippantonio et al, 2005; Zhong et al, 2005; Jazayeri et al, 2006; Myers and Cortez, 2006; Olson et al, 2007a). Many viruses interact with cellular DNA damage sensing and repair pathways (Lilley et al, 2007). In the case of human adenovirus (Ad), the cellular DNA repair machinery presents an obstacle to productive infection (Stracker et al, 2002). Infection with mutant Ad deleted of the E4 region results in formation of virus genome concatemers by a cellular DNA repair pathway (Boyer et al, 1999; Stracker et al, 2002). The MRN complex is required for concatemer formation, and wild-type Ad serotype 5 (Ad5) has evolved two ways to Figure 1 The Mre11 and Nbs1 proteins are required for robust ATR prevent concatemers by targeting the MRN complex signaling in response to mutant Ad infection. (A) Infections were (Stracker et al, 2002). The viral E1b55K and E4orf6 proteins performed in a transformed cell line deficient for Mre11 (A-TLD1) induce proteasome-mediated degradation of MRN proteins that was transduced with an empty retrovirus vector (vector) or a (Stracker et al, 2002; Carson et al, 2003), whereas expression vector expressing the wild-type Mre11 cDNA (Mre11). Cells were uninfected (Mock) or infected with wild-type Ad5 (Ad5) and the of the Ad5-E4orf3 protein mislocalizes MRN proteins into E4-deletion mutant (dl1004). Immunoblotting of lysates prepared at intranuclear track-like structures and cytoplasmic aggregates 30 hpi used antibodies for total protein (Chk1, RPA32, Mre11, Rad50 (Stracker et al, 2002, 2005; Araujo et al, 2005; Evans and and Nbs1) or phospho-specific sites (Chk1-S345). Ku86 served as a Hearing, 2005). loading control. (B) Infections were performed in the transformed NBS cell line (NBS-ILB1) transduced with an empty retrovirus Coincident with concatemer formation, infection with vector (vector) or a vector expressing the wild-type Nbs1 cDNA. E4-deleted Ad also elicits a cellular DNA damage response (Stracker et al, 2002; Carson et al, 2003). The ATM and ATR kinase signaling pathways are activated, as detected by autophosphorylation of ATM and phosphorylation of many versions of the A-TLD1 cell line that expresses mutant known ATM and ATR substrates (Carson et al, 2003). The Mre11 (Carson et al, 2003), and the NBS-ILB1 cell line that response is also characterized by recruitment of the MRN harbours an Nbs1 mutation (Cerosaletti et al, 2000). Lysates complex and other DNA damage response proteins to viral from cells infected with the E4-deleted virus dl1004 were replication centres. In contrast, this cellular DNA damage analyzed by immunoblotting with a phospho-specific anti- response is not observed during infection with wild-type Ad5 body to Chk1-S345, and with an antibody to RPA32 that expresses E4 proteins (Carson et al, 2003). Ad infection (Figure 1). Phosphorylation of Chk1 and RPA32 was detected provides a novel system to analyze the mechanism and in cell lines complemented with wild-type cDNAs. In con- consequences of ATR signaling. Here, we examine the effect trast, infection with E4-deleted Ad in the A-TLD1 (Figure 1A) of E4orf3-mediated MRN redistribution on ATM and ATR and NBS (Figure 1B) mutant lines produced significantly signaling. We show that activation of ATR signaling during reduced signaling. Wild-type Ad infection did not generate viral infection is dependent on the MRN complex but unlike these phosphorylation events due to degradation of MRN other types of damage is independent of ATM. The MRN proteins. These data show that the MRN complex is required complex is not required for accumulation of RPA, ATRIP, ATR for robust ATR signaling in response to Ad infection. and TopBP1 at viral replication centres. We examine MRN dynamics in living cells and show that E4orf3-induced im- The presence of Ad5-E4orf3 reduces ATR signaling but mobilization prevents MRN from responding to the virus and not ATM other types of DNA. These results show a novel role for MRN As the Ad5-E4orf3 protein affects MRN complex localization, in activation of ATR signaling independent of ATM and we investigated whether E4orf3 alters ATM and ATR signaling downstream of recruitment of RPA/ATR/ATRIP/TopBP1. during infection. Because the viral E1b55K and E4orf6 pro- teins downregulate damage signaling through degradation of the MRN complex (Carson et al, 2003), we tested a mutant Results that is deleted of E4orf6 and E1b55K but retains E4orf3 The MRN complex is required for robust ATR signaling (dl1017) (Bridge and Ketner, 1990), and compared it with during infection wild-type Ad5 and the large E4-deleted mutant (see We have showed earlier the phosphorylation of ATM and ATR Figure 2A). The E4-deleted virus induced phosphorylation kinase substrates in response to E4-deleted Ad infection of ATR substrates Chk1 and RPA32, as detected by a (Carson et al, 2003). Some sites were modified by either shift in the mobility of RPA32 and by recognition with ATM or ATR (e.g., Chk2-T68 and Nbs1-S343), whereas others phospho-specific antibodies (Chk1-S345 and RPA32-S4,8) were ATR specific (e.g., Chk1-S345 and RPA32). We showed (Figure 2B). These phosphorylation events were greatly that MRN is required for ATM activation (Carson et al, 2003), reduced during infection with dl1017 that expresses E4orf3, but the requirement for MRN in ATR signaling during virus indicating that ATR signaling is reduced in the presence of infection has not been clarified. To investigate the role of Ad5-E4orf3. MRN specifically in ATR-dependent signaling responses to We also examined the effect of E4orf3 on ATM signaling. In virus, we examined phosphorylation events during infections contrast to ATR, ATM signaling was intact during infection of cells with hypomorphic mutations in Mre11 and Nbs1. with dl1017, as evidenced by ATM autophosphorylation on Infections were performed in mutant and complemented S1981 (Figure 2B). Analysis of substrates that are phosphory- &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 653 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 2 Phosphorylation of ATR substrates is reduced in the presence of Ad5-E4orf3 and is independent of ATM. (A) Genotypes of mutant viruses used for infection (Bridge and Ketner, 1990). (B) HeLa cells were mock infected or infected with indicated viruses. Cells were harvested at 6, 12, 18 and 24 hpi and prepared for analysis by immunoblotting using antibodies to phospho-specific residues (Chk1-S345, RPA32-S4,8 and ATM-S1981). Below are lysates at 24 hpi probed with antibodies against total protein (Chk1, RPA32 and Ku86) to serve as loading controls. (C) Immunoblots of infections in control cells (48BR) or Seckel cells with mutant ATR (GM18366). (D) Immunoblots of infections in A-T cells with mutant ATM (AT221JE-T) or a complemented line (A-T þATM). DBP was a marker of virus infection and Ku86 served as a loading control. lated by both ATM and ATR (Chk2-T68, Nbs1-S434 and Accumulation of ATR at viral replication centres is BRCA1) revealed that their phosphorylation was decreased independent of MRN but not abolished by E4orf3 expression (Supplementary Viral replication centres can be detected by staining with Figure S1A), showing that E4orf3 inhibits ATR but not antibodies to the viral DNA-binding protein (DBP) that coats ATM. Immunofluorescence during infection with dl1017 ssDNA accumulated at viral replication sites (Pombo et al, showed that MRN mislocalization by E4orf3 did not prevent 1994). The cellular RPA32 protein also accumulates at these ATM activation, and that phosphorylated Nbs1 colocalized sites (Stracker et al, 2005), with both wild-type and mutant with Rad50 in nuclear tracks (Supplementary Figure S1B). viruses (Figure 3A). The E4orf3 protein is excluded from viral Together, these data suggest that MRN mislocalization by replication centres and appears in intranuclear tracks and E4orf3 is not sufficient to prevent ATM activation, indicating cytoplasmic aggregates (Araujo et al, 2005; Evans and that ATM and ATR signaling pathways can be independently Hearing, 2005), with both wild-type Ad5 or the dl1017 mutant regulated during Ad infection. (Figure 3A). The Ad5-E4orf3 protein is sufficient to redistri- To determine the interdependence of ATR and ATM, we bute the MRN complex, as detected by staining for Nbs1 examined signaling in cells with kinase mutations. Cells (Figure 3A). In contrast, infection with dl1004 leads to derived from a Seckel syndrome patient with mutation in accumulation of MRN at viral centres. We, therefore, inves- ATR (O’Driscoll et al, 2003) and from an A-T patient with tigated whether MRN mislocalization by E4orf3 correlates mutation in ATM were infected with viruses that express with inhibition of ATR signaling (Figure 3B). Phosphorylation E4orf3 (Ad5 or dl1017) and those that lack E4orf3 (dl1004 or of RPA32 at viral centres, as detected by staining with the dl1016). Phosphorylation of Chk1-S345 and RPA32-S4,8 was RPA32-S4,8 antibody, was only observed when the MRN abolished in the Seckel cells (Figure 2B). ATM activation complex was associated with viral replication centres (i.e., and signaling were intact in Seckel cells, as detected by in the absence of E4orf3 during infection with dl1004). When autophosphorylation on S1981 (Figure 2C) and phosphory- the virus expressed E4orf3, RPA32 phosphorylation was not lation of downstream substrates (Supplementary Figure S1C). detected. This shows that E4orf3 mislocalization of the MRN Infections of A-T cells or a matched complemented line with complex prevents its accumulation at viral replication cen- the mutant virus dl1016 that lacks both E1b55K and E4orfs tres, and that this correlates with the absence of ATR signal- 1-3 (DE1b55K/DE4orf1-3) induced ATR signaling (Figure 2C). ing to RPA32. However, signaling to Chk1 and RPA32 was abrogated in the We also investigated whether E4orf3 affects localization of presence of E4orf3 (dl1017). These results show that ATR other proteins involved in ATR signaling. We observed that activation in response to Ad infection, and its inhibition by ATR, RPA and ATRIP accumulated at viral replication centres E4orf3, are independent of ATM. with all viruses tested, irrespective of E4orf3 expression 654 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 3 Signaling by ATR at viral replication centres. (A) E4orf3 sequesters Nbs1 into tracks away from viral replication centres. HeLa cells were mock infected or infected with indicated viruses. Cells were fixed at 16 hpi, and immunofluorescence was performed. Staining shows that Nbs1 but not RPA32 is excluded from viral replication centres in the presence of E4orf3. (B) Colocalization of the MRN complex with RPA32 at replication centres correlates with RPA32 phosphorylation. Immunofluorescence with a phospho-specific antibody to RPA32-S4,8 shows colocalization with viral replication centres stained with an antibody to DBP only in the absence of E4orf3. (C) Accumulation of RPA32, ATR and ATRIP at viral centres is independent of E4. (D) TopBP1 accumulates at viral centres independently of E4. (E) Activation is observed for ATM, but not ATR, in Seckel cells. (F) ATR signaling is restored in A-TLD cells by expression of Mre11 or the nuclease-defective mutant Mre11-3. In all images, the nuclei were located by co-staining cellular DNA with DAPI (blue). (Figure 3C) (Carson et al, 2003; data not shown). These data activity through an interaction with ATRIP (Kumagai et al, suggest that E4orf3 does not abrogate ATR signaling by 2006; Mordes et al, 2008). We examined localization of mislocalizing ATR, RPA32 or ATRIP. It also shows that the TopBP1 during infection and found it localized in viral DBP presence of ATR at viral centres is not sufficient to initiate or centres with wild-type and E4 mutant viruses (Figure 3D). maintain the DNA damage signaling during infection. The Immunofluorescence of infected Seckel cells confirmed that TopBP1 protein is a regulator of ATR that stimulates kinase phosphorylation of RPA32-S4,8 was not observed in the &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 655 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al absence of functional ATR, although RPA32 and ATRIP basis of these alignments, we converted isoleucine at position accumulated at viral centres (Figure 3E). Lack of RPA32 104 of Ad5 to arginine, which is present in all other serotypes phosphorylation was confirmed in Seckel cells from multiple (Figure 4A). The Ad5 E4orf3-I104R mutant retained the origins and could also be restored by introduction of the wild- ability to form nuclear tracks and disrupt PML but was type ATR gene (Supplementary Figure S2). ATM activation defective for redistribution of the MRN complex was detectable in Seckel cells as revealed by staining for (Figure 4B). This identified a region of E4orf3 involved in ATM-S1981 and is therefore independent of ATR in response targeting the MRN complex and provided a separation-of- to Ad infection (Figure 3E). Although RPA and ATR were function mutant. detected at viral centres in the absence of functional MRN We next examined the ability of Ad5-E4orf3, Ad12-E4orf3 complex, phosphorylation of RPA32 was not observed and E4orf3-I104R proteins to prevent ATR signaling during during infections of NBS and A-TLD1 cells (Figure 3F, infection with E4-deleted virus (Figure 5). The cells were Supplementary Figure S2C and D). ATR signaling was re- transfected with E4orf3 expression vectors, and then infected stored when infections were performed in A-TLD1 cells with dl1004. Immunoblotting revealed abrogated ATR signal- transduced with retroviruses that express either wild-type ing in the presence of Ad5-E4orf3 (reduced Chk1-S345 and Mre11 or the nuclease-defective Mre11-3 mutant (Figure 3F). RPA32-S4,8 phosphorylation and RPA32 mobility shift), Together, these data show that the MRN complex is not although ATM autophosphorylation was not significantly required for localization of ATR with ssDNA at viral replica- altered (Figure 5A). In contrast, the Ad12-E4orf3 and tion centres but is required for ATR signaling. E4orf3-I104R proteins, which failed to mislocalize the MRN complex, did not inhibit ATR signaling (Figure 5A and B). In Mislocalization of the MRN complex by Ad5-E4orf3 is this experiment, there was a slight decrease in ATM signaling required for disruption of ATR signaling (Figure 5B), but this was not reproducible and did not Although E4orf3 proteins from all serotypes tested form correlate with MRN mislocalization. Cells from the same nuclear tracks, rearrange the promyelocytic leukemia protein experiment were also examined by immunofluorescence for RPA32-S4,8 phosphorylation, as a marker of ATR signaling PML and redistribute components of the PML bodies, only Ad5-E4orf3 mislocalizes the MRN complex (Stracker et al, (Figure 5C). We observed that Ad5-E4orf3, but not Ad12- 2005). Sequence analysis for the highly conserved E4orf3 E4orf3 or E4orf3-I104R, prevented RPA phosphorylation at proteins revealed residues specific to the subgroup C viruses viral replication centres. Cotransfection of a plasmid expres- (Ad1, Ad2 and Ad5) that uniquely mislocalize MRN. On the sing GFP served as a marker for transfected cells. Control Figure 4 A region in Ad5-E4orf3 important for targeting the MRN complex. (A) Sequence alignment of E4orf3 proteins. E4orf3 sequences from different human (subgroup noted in brackets) and simian adenoviruses were aligned using the CLUSTAL algorithm and the region around residue I104 is shown. Conserved residues are shown boxed, with CLUSTAL colour scheme reflecting amino acids of similar chemical nature. The I104 residue is highlighted, showing that this site differs between subgroup C and all other sequenced E4orf3 genes. (B) The Ad5 E4orf3- I104R mutant does not redistribute the MRN complex. Plasmids for wild-type and mutant E4orf3 were transfected into HeLa cells. Immunofluorescence shows that the E4orf3 mutant protein still forms tracks and disrupts PML structures but is unable to redistribute members of the MRN complex, which remain diffusely nuclear. Representative images are shown and nuclei are located by co-staining cellular DNA with DAPI. 656 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al structures and accumulated in cytoplasmic aggregates (Figure 6A). Both Mre11–YFP and Nbs1–YFP fusion protein colocalized with endogenous Rad50 protein. Similar results were obtained using a stable cell line expressing Nbs1–2GFP (Lukas et al, 2003) (data not shown). These results show that the fusion proteins are correctly incorporated into E4orf3- induced structures. During infection with E4-deleted virus, Nbs1–YFP formed foci at viral replication centres (Figure 6A), as reported earlier for endogenous MRN (Stracker et al, 2002). We then examined the dynamics of MRN in E4orf3 tracks using fluorescence recovery after photobleaching (FRAP) to determine the rate at which a bleached area is repopulated with new fluorescent protein. As reported earlier, we ob- served that the Mre11 and Nbs1 proteins are highly mobile in the absence of E4orf3 (Figure 6B). FRAP analysis of Nbs1 foci at viral replication centres during infection with E4-deleted virus dl1004 showed that the signal recovered with kinetics slower than that of an undamaged area (Figure 6C). The increased residence time at virus centres resembles that observed for recruitment of the MRN complex at DSBs (Lukas et al, 2003). In contrast, the kinetics of fluorescence recovery was much more significantly affected by the pre- sence of E4orf3, where the fluorescence signal did not recover after photo-bleaching of MRN in tracks induced by E4orf3 during transfection (Figure 6B) or infection (Figure 6C). We also compared MRN mobility in the presence of wild-type and I104R mutant E4orf3 proteins (Figure 6D). Although wild-type Ad5-E4orf3 expression led to immobilization of Mre11–YFP, the I104R mutant barely affected Mre11 dy- namics. This supports our observations from fixed images that show I104R does not alter Mre11 localization. Together, Figure 5 ATR signaling in response to virus infection is abrogated these data show that MRN in tracks induced by Ad5-E4orf3 by E4orf3 proteins that mislocalize the MRN complex. HeLa cells represent immobilized proteins. were transfected with an empty plasmid (vector) or with vectors expressing E4orf3 proteins. After 24 h, cells were either mock E4orf3 prevents ATR-dependent signaling in response infected, infected with the E1b55K/E4orf6 mutant (dl1017) (positive control), or infected with the E4-deletion mutant (dl1004). (A, B) to nonviral damage Lysates from cells at 24 hpi were immunoblotted with antibodies to To assess the impact of MRN immobilization on the ability of Chk1-S345, RPA32-S4,8, ATM-1981 and RPA32. (C) Cells were fixed cells to respond to DNA damage, we generated spatially at 18 hpi, and immunofluorescence was performed with an anti- restricted DSBs through laser microirradiation (Lukas et al, body to RPA32-S4,8. Images are shown merged with DAPI staining. A plasmid expressing GFP was cotransfected with that for E4orf3 at 2003). Local DNA damage was generated in subnuclear a ratio of 1:10 and GFP staining is shown in the lower left insert regions by a focussed laser beam programmed to move panel as a positive control for transfection. once across individual cell nuclei. Previous studies using visualization with specific antibodies and fluorescently tagged proteins have shown that DNA damage proteins are staining showed that viral DBP centres were formed in all redistributed to these DSBs (Lukas et al, 2003, 2004). We cells, and that expression of E4orf3 proteins resulted in PML examined recruitment of endogenous cellular repair proteins disruption (data not shown). Together, these data support the to laser-induced DSBs in cells expressing E4orf3. The sites of hypothesis that mislocalization of the MRN complex by microirradiation can be visualized by recruitment of the E4orf3 leads to abrogation of ATR signaling. mediator protein Mdc1 (Lukas et al, 2004), which is unaf- fected by E4orf3 (Figure 7A). Cells expressing Ad5-E4orf3 The MRN complex is immobilized by Ad5-E4orf3 localized endogenous Nbs1 into the characteristic nuclear We reported earlier that Ad5-E4orf3 expression alters MRN tracks and displayed minimal accumulation of Nbs1 at micro- complex solubility (Araujo et al, 2005). To examine the effect irradiation sites compared with neighbouring untransfected of E4orf3 on protein dynamics in live cells, we used fluores- cells. This result shows that Nbs1 immobilization in E4orf3- cently tagged fusion proteins of Mre11 and Nbs1. We ex- induced tracks prevents its accumulation at DSBs. pressed Mre11–YFP and Nbs1–YFP by cell transfection either To examine the effect of E4orf3 expression on kinetics of in the presence of E4orf3 alone or in virus-infected cells the DNA damage response, we combined laser microirradia- (Figure 6). The fusion proteins were distributed diffusely in tion with live cell imaging (Figure 7B). The Nbs1–2GFP cell the nucleoplasm in untreated cells (Lukas et al, 2003). In the line was cotransfected with the Ad5-E4orf3 expression vector presence of E4orf3, provided by virus infection or plasmid and a plasmid expressing RFP to serve as a transfection transfection, the fusion proteins localized into track-like marker. Generation of subnuclear restricted DSBs resulted &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 657 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 6 E4orf3 abrogates MRN function by immobilizing the MRN complex. (A) HeLa cells were transfected with Nbs1–YFP or Mre11–YFP alone or together with Ad5-E4orf3 plasmid vector. After 24 h, cells were infected with dl1004 or dl1017. Cells were fixed 18 hpi, and immunofluorescence was performed with a Rad50 antibody. Images shown are merged with DAPI staining. (B–D) FRAP analysis of Nbs1 and Mre11 in cells expressing E4orf3 by infection or transfection. The unbleached portion of the cell served to normalize the overall fluorescence decay during the repeated image collection. Arrows above indicate the time of bleaching. (B) Stable U2OS cells expressing Nbs1–2GFP were either untreated, transfected with E4orf3, or treated with 10 Gy gamma-irradiation. (C) HeLa cells were transfected with Nbs1–YFP and then mock treated or infected with dl1004 and dl1017. Cells were analyzed by FRAP at 18 hpi. (D) HeLa cells transfected with Mre11–YFP together with either empty vector or vectors expressing Ad5-E4orf3 and E4orf3-I104R were analyzed by FRAP. in rapid recruitment of Nbs1–2GFP. In contrast, there was report, we show that MRN also has an important function barely detectable recruitment of Nbs1 in cells expressing in ATR signaling in response to E4-deleted Ad. Substrates Ad5-E4orf3, even at 15 min after treatment. These data known to contribute to ATR kinase activation include ssDNA indicate that the MRN complex immobilized in E4orf3- coated with RPA, and junctions of single- and double- induced tracks is unable to respond efficiently to exogenous stranded DNA (MacDougall et al, 2007; Zou, 2007). Our DNA damage. As MRN has been shown to be required for data show that ssDNA at viral replication centres (Pombo ATR signaling in response to HU treatment (Manthey et al, et al, 1994) is sufficient for recruitment of ATR and ATRIP, 2007), we used recombinant Ad vectors to express but that ATR signaling to Chk1 and RPA32 is only detected Ad5-E4orf3 or GFP in cells and then exposed them to HU when the MRN complex also accumulated at viral centres. (Figure 7C). Signaling was abrogated by E4orf3 as shown by Although we have focussed on MRN, we cannot exclude decreased hyperphosphorylation of RPA32 and staining additional roles for other proteins implicated in ATR activa- with phospho-specific antibodies to RPA32-S4,8, Nbs1-S343 tion and checkpoint signaling, such as the 9-1-1 complex, the (Figure 7C), and Chk1-S345 (data not shown). Together, these Rad17 complex and Claspin (Zou, 2007). The cellular data show that immobilization of MRN by E4orf3 prevents E1b55K-associated protein E1B-AP5 was recently implicated the ATR-mediated response to replication stress. in ATR signaling during Ad infection (Blackford et al, 2008), but this factor localizes to wild-type Ad5 centres and, there- fore, does not explain the induction of ATR signaling in the Discussion absence of E4. The role of MRN in ATR activation MRN has recently been implicated in facilitating ATR The MRN complex has an important function in the cellular activation and signaling in response to some types of damage. DNA repair response to Ad infection. We showed earlier that Processing of DSBs in an MRN-dependent manner results in the formation of ssDNA and ATR activation (Adams et al, during infection with E4-deleted Ad, MRN is required for 2006; Jazayeri et al, 2006; Myers and Cortez, 2006). MRN concatemerization of the viral genome and activation of ATM is involved in ATR-mediated phosphorylation events in signaling (Stracker et al, 2002; Carson et al, 2003). In this 658 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al Figure 7 E4orf3 prevents ATR-dependent damage signaling induced by nonviral sources. (A) U2OS cells transfected with a plasmid vector expressing Ad5-E4orf3 were laser microirradiated. Cells were fixed and stained for endogenous Nbs1 and Mdc1. Arrows indicate cells with E4orf3-induced tracks of Nbs1. (B) Stable U2OS cell lines expressing Nbs1–2GFP were transfected with a plasmid expressing Ad5-E4orf3, together with prRFP-C1, which was used as a marker for transfected cells. Cells were laser microirradiated (as indicated by dashed line) and images show the recruitment of Nbs1 to sites of damage. (C) HeLa cells were mock infected, or infected with E1-deleted recombinant Ads expressing GFP (rAd-GFP) or E4orf3 (rAd-E4orf3). At 24 hpi, the cells were mock treated or treated with 2 mM hydroxyurea (HU) for 2 h, and the cells were harvested for immunoblotting. Cellular proteins were detected with antibodies to RPA32 and specific phosphorylated sites at RPA-S4,8 and Nbs1-S343. Ku86 served as a loading control. response to replication stress, although signaling events may point (Robison et al, 2004; Olson et al, 2007b) and also also be MRN independent, depending on the substrate and interacts with ATR/ATRIP (Olson et al, 2007a). Either of dose of damaging agent (Pichierri and Rosselli, 2004; Stiff these two interactions could contribute to ATR activation by et al, 2005; Zhong et al, 2005; Olson et al, 2007b). During MRN at viral centres. The ATM and ATR kinases may be infection with E4-deleted Ad, it is not clear which DNA coordinated and interdependent in response to some types of structures serve as the trigger for ATR signaling, and there damage (Hurley and Bunz, 2007). In response to IR, activa- may be multiple ways that the MRN complex contributes to tion of ATR is ATM dependent (Jazayeri et al, 2006; Myers ATR activation. Although the nuclease activity of Mre11 is and Cortez, 2006), whereas in response to HU and UV required for joining Ad genomes into concatemers (Stracker activation of ATM is ATR dependent (Liu et al, 2005; Stiff et al, 2002), we found that it was not required for ATR et al, 2006). In the case of virus infection, we have found that signaling. In contrast to DSBs caused by IR or replication although both rely upon the MRN complex, ATM and ATR stress, where the nuclease activity of Mre11 is required for signaling are independent of each other. This may reflect the resection (Buis et al, 2008), we found that Mre11 nuclease fact that during infection there are numerous substrates for activity is not required for generation of ssDNA and recruit- kinase activation, including replication intermediates and ment of RPA/ATR/ATRIP at viral centres. However, signaling double-strand ends. In addition to the viral genome, infection may be triggered by further processing of the viral genome, may also induce chromosomal damage to the host genome. for example, by removal of the terminal protein from the 5 Early Ad genes alter cell-cycle progression, which could lead end of the genome by other nucleases to generate free DNA to collapse of replication forks, and also cause genomic ends. MRN-dependent processing of DSBs has been sug- instability and chromosomal aberrations (Caporossi and gested to generate small oligonucleotides that stimulate Bacchetti, 1990; Lavia et al, 2003). Therefore, although the ATM activity (Jazayeri et al, 2008). It will be interesting to phosphorylated ATR substrates predominantly accumulate at determine whether these are generated during virus infection viral replication centres, it is possible that chromosomal and whether they play a role in ATR activation. The MRN damage also contributes to induction of ATR signaling during complex could have a direct role in stimulating ATR kinase infection. activity, as has been shown in vitro for ATM (Lee and Paull, 2004). The MRN complex may also facilitate phosphorylation The function of viral E4 proteins of downstream substrates through recruitment or retention of There is functional redundancy between the E4orf3 and proteins to viral centres, as has been proposed for stalled E4orf6 products from Ad, and either is sufficient to promote replication forks (Stiff et al, 2005). MRN associates with RPA viral replication, prevent concatemerization of the viral at sites of DNA damage to mediate the intra-S-phase check- genome, and enable viral late protein production. Both E4 &2009 European Molecular Biology Organization The EMBO Journal VOL 28 NO 6 2009 659 | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al proteins target the MRN complex to prevent concatemer (Wilkinson and Weller, 2006). Induction of the Epstein–Barr formation (Stracker et al, 2002; Evans and Hearing, 2003). virus lytic program also elicits a cellular DNA damage re- Together with previous observations of MRN degradation by sponse and ATM activation, but ATR signaling is minimal E1b55K/E4orf6 (Carson et al, 2003), our data show that both (Kudoh et al, 2005). Therefore, viruses appear to use multiple E4 proteins target MRN to prevent damage signaling. strategies to inactivate ATR and downstream signaling events Inactivation of MRN is also likely to be responsible for the during viral infection, to prevent negative impacts of the ability of E4 proteins to promote viral DNA replication. We cellular response to replication stress on virus production. and others have found that MRN inhibits replication of E4- Understanding how viruses such as Ad manipulate signaling deleted mutant Ad, although the mechanism is unclear pathways will provide insights into the regulation of DNA (Evans and Hearing, 2005; Mathew and Bridge, 2007, 2008; damage responses in mammalian cells. Lakdawala et al, 2008). Replication of cellular DNA is tightly regulated to ensure that the genome is replicated only once Materials and methods per cell cycle (Arias and Walter, 2007). MRN is recruited to cellular replication origins and can inhibit firing of new Cell lines origins of DNA replication upon damage (Olson et al, HeLa and 293 cells were purchased from the American Tissue 2007b) and suppress rereplication (Wu et al, 2004; Lee Culture Collection. W162 cells for growth of E4-deleted viruses were from G Ketner, A-T cells (AT221JET and complemented et al, 2007). Mre11 has recently been suggested to bind the version) were from Y Shiloh, and Seckel cells were from Coriell Ad genome (Mathew and Bridge, 2008), but it is unclear how Institute (GM18366) and A D’Andrea (F02-98). Immortalized this inhibits replication. Checkpoint signaling by ATM and A-TLD1 and NBS (NBS-ILB1) and matched cells reconstituted with ATR is not responsible for the defective replication of E4- wild-type Mre11 and NBS1 were described (Cerosaletti et al, 2000; Carson et al, 2003). The retrovirus expression plasmid for the deleted Ad (Lakdawala et al, 2008). The virus uses its own Mre11-3 mutant (HD129/130LV) was generated by site-directed protein-priming mechanism and polymerase, which could be mutagenesis and A-TLD1 cells were transduced by the retrovirus as affected by MRN binding to the origin or its participation in described previously (Carson et al, 2003). The U2OS-derived stable removal of the terminal protein from the viral genome. cell line with Nbs1–2GFP has been described (Lukas et al, 2003). Cells were maintained as monolayers in either Dulbecco modified Our work links the E4orf3-induced redistribution of pro- Eagle’s medium (DMEM) or MEM plus Earle’s salts (Seckel cells) teins associated with PML nuclear bodies to their role in supplemented with 10 or 20% fetal bovine serum (FBS), at 371Cin a sensing DNA damage (Everett, 2006). We show that reloca- humidified atmosphere containing 5% CO . lization of the MRN complex dramatically reduces its dynamics, essentially immobilizing the proteins in E4orf3- Plasmids and transfections induced intranuclear tracks. Similar observations were made Expression vectors for Ad5-E4orf3 and Ad12-E4orf3 proteins were with FRAP analysis of other fluorescently tagged components described (Stracker et al, 2005). Site-directed mutagenesis of E4orf3 was performed using QuikChange (Stratagene). Cells were trans- of the PML bodies (unpublished observations). Experiments fected with Lipofectamine 2000 (Invitrogen) according to manu- with laser microirradiation and live cell imaging showed that facturer’s protocol. recruitment of MRN to damage sites was severely abrogated in cells expressing Ad5 E4orf3. This correlated with decreased Viruses and infections recruitment of RPA and ATR, as well as abrogated damage The mutant viruses dl1004 (DE4), dl1016 (DE4orf1-3/DE1b55K) and signaling, and was not seen with the I104R mutant (data not dl1017 (DE4orf6/DE1b55K) have been described (Bridge and Ketner, 1990) and were obtained from G. Ketner. Wild-type Ad5 and dl1017 shown). Together, these results show that immobilization by were propagated in 293 cells. The dl1004 and dl1016 viruses were E4orf3 prevents the MRN damage sensor from responding to propagated on W162 cells (Weinberg and Ketner, 1983). All viruses new damage sites. This supports the hypothesis that seques- were purified by two sequential rounds of ultra-centrifugation in tering MRN (and other host factors) into E4orf3-induced cesium chloride gradients and stored in 40% glycerol at 201C. Infections were performed in DMEM supplemented with 2% FBS. tracks will prevent these proteins from sensing and accumu- After 2 h at 371C additional serum was added to a total of 10%. lating at virus centres and will thus thwart host antiviral responses. It has also been suggested that sequestration of Antibodies MRN in cytoplasmic aggresomes by the adenoviral E1b55K Primary antibodies were purchased from Novus Biologicals Inc. inactivates the complex and protects the viral genome (Liu (Nbs1), Genetex (Mre11-12D7, Rad50-13B3), Cell Signaling (Chk1- et al, 2005). As the E4orf3 and E4orf6 proteins both block S345), Rockland (ATM S1981-P), Santa Cruz (ATR, PML, Chk1, Ku86), Upstate Biotechnology (ATRIP), BD Bioscience (TopBP1) damage signaling and also promote production of late viral and Bethyl (RPA32-S4,8). The antibody to RPA32 was from proteins, it will be interesting to determine whether these T. Melendy, the monoclonal B6 antibody to DBP was from activities are linked. A. Levine, polyclonal rabbit antisera to DBP was from P. van der Inactivation of ATR may be a general approach used by Vliet, and the E4orf3 antibody was from T. Dobner. Secondary antibodies were from Jackson Laboratories, Eurogentec and viruses to neutralize aspects of the host defense. In addition Invitrogen Molecular Probes. to our observations with Ad infection, it has been suggested that ATR signaling is manipulated by other DNA viruses. For Immunoblotting and immunofluorescence example, in the case of herpes-simplex virus type 1 (HSV-1), Immunoblotting and immunofluorescence were performed as infection activates ATM signaling pathways and results in described previously (Carson et al, 2003). Novex (Invitrogen) 3 to accumulation of cellular repair proteins at viral centres (Lilley 8% gradient gels were used for the resolution of ATM. For et al, 2007). Although RPA is found at HSV-1 viral replication immunofluorescence, cells grown on glass coverslips were infected at an MOI of 25–100 pfu/cell. After 16–24 h, the cells were washed, compartments, ATR-dependent signaling is not activated, and fixed, stained and counter-stained with 4 ,6-diamidino-2-phenylin- it has been suggested that it is prevented because of spatial dol (DAPI). Immunoreactivity was visualized using a Nikon uncoupling of the ATR-ATRIP complex and sequestering of microscope in conjunction with a CCD camera (Cooke Sensicam) phosphorylated RPA in viral-induced nuclear domains or a Leica confocal microscope. 660 The EMBO Journal VOL 28 NO 6 2009 &2009 European Molecular Biology Organization | | Adenovirus E4orf3 prevents ATR signaling CT Carson et al FRAP analysis reagents. We thank members of the Weitzman lab, past and present, FRAP analysis was performed in the Nbs1–2GFP U2OS cells as for discussions and critical reading of the manuscript. We thank D reported previously (Lukas et al, 2003). Further details are provided Ornelles for discussions and sequence analysis for E4orf3. We in Supplementary data. Image collection and FRAP data were acknowledge the James B Pendleton Charitable Trust for providing processed on a Leica confocal microscope. the Pendleton Microscopy Facility. This work was supported by NIH grant CA97093 (MDW), and by gifts from the Joe W & Dorothy Supplementary data Dorsett Brown Foundation and the Lebensfeld Foundation to MDW. Supplementary data are available at The EMBO Journal Online Additional support came from The Danish National Research (http://www.embojournal.org). Foundation, Danish Cancer Society, European Commission (DNA Repair) and the John and Birthe Meyer Foundation. CTC was supported by the Timken-Sturgis Foundation and a scholarship Acknowledgements from the ARCS Foundation. CTC and NIO were supported in part by an NIH Training Grant to the Salk Institute. 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Journal

The EMBO Journal – Springer Journals

Published: Mar 18, 2009

Keywords: adenovirus; ATR kinase; DNA damage response; MRN complex

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