Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation Andrew N. Blackforda,1,2, Rakesh N. Patela, Natalie A. Forrestera, Kathrin Theilb, Peter Groitlb, Grant S. Stewarta, A. Malcolm R. Taylora, Iain M. Morganc, Thomas Dobnerb, Roger J. A. Granda,2, and Andrew S. Turnella,2 aSchool of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom; bHeinrich Pette Institute for Experimental Virology and Immunology, 20251 Hamburg, Germany; and cInstitute of Comparative Medicine, University of Glasgow Faculty of Veterinary Medicine, Glasgow G61 1QH, United Kingdom Edited* by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved May 14, 2010 (received for review December 22, 2009) Activation of the cellular DNA damage response is detrimental to Adenovirus type 5 (Ad5) is a linear dsDNA tumor virus that adenovirus (Ad) infection. Ad has therefore evolved a number of provides a useful model system for studying the DNA damage strategies to inhibit ATM- and ATR-dependent signaling pathways response, as ATM and ATR signaling pathways are inhibited during infection. Recent work suggests that the Ad5 E4orf3 protein during infection (11). Ad5 E1B-55K and E4orf6 proteins prevent prevents ATR activation through its ability to mislocalize the MRN ATM activation by promoting the ubiquitin-dependent protea- complex. Here we provide evidence to indicate that Ad12 has somal degradation of MRN (11, 12). Cullin-RING ubiquitin evolved a different strategy from Ad5 to inhibit ATR. We show that ligases (CRLs) are multiprotein complexes required for the pol- Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to yubiquitylation and proteasomal degradation of a large number promote the proteasomal degradation of the ATR activator protein of cellular proteins, to control many diverse processes such as cell topoisomerase-IIβ–binding protein 1 (TOPBP1). Ad12 also uses this cycle progression, limb patterning, and glucose sensing (13). complex to degrade p53 during infection, in contrast to Ad5, which During Ad5 infection, E1B-55K and E4orf6 associate with a CRL requires a CUL5-based ubiquitin ligase. Although Ad12-mediated complex containing CUL5, RBX1, and elongins B and C, and degradation of p53 is dependent upon both E1B-55K and E4orf6, serve to target this CRL to new substrates (14, 15). It has been Ad12-mediated degradation of TOPBP1 is solely dependent on suggested that E1B-55K is the substrate receptor for the complex, E4orf6. We propose that Ad12 E4orf6 has two principal activities: and E4orf6 is the adaptor that recruits the CRL as it has several to recruit the CUL2-based ubiquitin ligase and to act as substrate motifs, termed BC boxes, that bind to the elongin B and C sub- receptor for TOPBP1. In support of the idea that Ad12 E4orf6 spe- units (16, 17). As virtually all detectable Ad5 E1B-55K/E4orf6 fi ci cally prevents ATR activation during infection by targeting associates with the CUL5 complex (15), it is likely that targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can in- cellular proteins for degradation is the major function of E1B- hibit the ATR-dependent phosphorylation of CHK1 in response to 55K and E4orf6 during Ad5 infection (18). replication stress. Taken together, these data provide insights into E1B-55K/E4orf6–dependent degradation of MRN is sufficient how Ad modulates ATR signaling pathways during infection. to prevent ATM activation (11), and this appears to be conserved between some Ad species (19). Interestingly, however, two viral DNA damage | cullin-RING ubiquitin ligases | DNA damage | proteasome serotypes, Ad5 and Ad12, differentially regulate ATR during in- fection (19). The Ad5 E4orf3 protein inhibits ATR activation by aintenance of genome integrity is essential for cell survival. relocalizing and immobilizing MRN subunits before their tar- MCells have therefore evolved a complex set of biochemical geted degradation by E1B-55K/E4orf6 (20), but Ad12 E4orf3 pathways to recognize and repair damaged DNA (1). In addition lacks this activity (21). Yet both viruses inhibit CHK1 phosphor- to recognizing cellular DNA lesions, there is increasing evidence ylation, indicating that full ATR activation is prevented by both to show that this response is also activated by viral DNA during Ad5 and Ad12, but by different strategies (19). infection, and that some DNA repair proteins can inhibit viral Here, we provide evidence to show that a virus can inhibit ATR replication (2). signaling by specifically targeting its activator protein, TOPBP1. The DNA damage response is primarily controlled by two re- Ad12 E4orf6 assembles a CRL complex that targets TOPBP1 for lated protein kinases, ataxia-telangiectasia mutated (ATM) and proteasomal degradation. In contrast to previous work with Ad5, ATM/Rad3-related (ATR). Both kinases have a large and partially we show that this CRL contains CUL2 rather than CUL5, and overlapping set of substrates that includes tumor suppressor and that Ad12 E1B-55K is not required for TOPBP1 degradation. checkpoint proteins such as p53 and BRCA1 (3). However, some Rather, Ad12 E4orf6 interacts directly with TOPBP1 and is proteins, such as the checkpoint effector kinases CHK1 and CHK2, necessary and sufficient to promote its degradation. Finally, we MICROBIOLOGY are specific substrates for ATR and ATM, respectively (4). ATM show that, in uninfected cells exposed to replication stress, ex- predominantly responds to DNA double-strand breaks (DSBs), ogenous expression of Ad12 E4orf6 prevents CHK1 phosphory- which are sensed by the MRE11-RAD50-NBS1 (MRN) complex lation, indicating that by targeting TOPBP1 for degradation, (5). MRN is required for ATM activation, as it stimulates both ATM recruitment to DSBs and phosphorylation of its substrates (5, 6). In contrast, ATR becomes active in response to a much Author contributions: A.N.B., R.J.A.G., and A.S.T. designed research; A.N.B., R.N.P., N.A.F., wider range of genotoxic stresses (4). This is probably accomplished R.J.A.G., and A.S.T. performed research; K.T., P.G., G.S.S., A.M.R.T., I.M.M., and T.D. con- tributed new reagents/analytic tools; A.N.B., R.J.A.G., and A.S.T. analyzed data; and A.N.B., by the recognition of a common signal, ssDNA, which can be R.J.A.G., and A.S.T. wrote the paper. generated at stalled replication forks or by enzymatic processing of The authors declare no conflict of interest. DNA lesions (4). Replication protein A (RPA) binds to tracts of *This Direct Submission article had a prearranged editor. ssDNA and recruits ATR by interacting with its essential binding 1 partner, ATR-interacting protein (ATRIP) (7, 8). ATR activation Present address: Weatherall Institute of Molecular Medicine, University of Oxford, John β– Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom. also requires other proteins, including topoisomerase-II binding 2 To whom correspondence may be addressed. E-mail: [email protected], protein 1 (TOPBP1) (9, 10). TOPBP1 contains a domain that [email protected], or [email protected]. stimulates ATR kinase activity, and so is essential for the phos- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. phorylation of many ATR targets, including CHK1 (9, 10). 1073/pnas.0914605107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0914605107 PNAS | July 6, 2010 | vol. 107 | no. 27 | 12251–12256 Downloaded by guest on September 24, 2021 E4orf6 can directly inhibit ATR in the absence of other viral pathway are recruited to Ad5 and Ad12 viral replication centers in proteins. Taken together, this study establishes Ad12 E4orf6 as infected cells (11, 19–21). Given the apparent differences in ATR a modulator of CRL-dependent ubiquitylation and a regulator of and TOPBP1 regulation in Ad5- and Ad12-infected cells, we ex- ATR/TOPBP1-dependent signaling. amined TOPBP1 localization patterns in these cells. In mock- treated cells, both RPA32 and TOPBP1 were found to be diffusely Results localized in the nucleoplasm, but excluded from nucleoli (Fig. TOPBP1 Is Degraded in Ad12- but Not Ad5-Infected Cells. Ad5 E1B- 2A). However, in Ad5-infected cells, TOPBP1 was relocalized to 55K and E4orf6 proteins promote proteasomal degradation of sites of viral replication, where it colocalized with Ad5 DNA- MRN to inhibit ATM signaling (11). We reasoned that Ad12 might binding protein (DBP), E1B-55K, and RPA32 at early and late also degrade one or more other cellular proteins to inhibit ATR. times after infection (Fig. 2B and Fig. S1). Next, we determined Therefore, we infected HeLa cells with WT Ad5 or Ad12 and TOPBP1 localization in Ad12-infected cells, using RPA32 as examined the steady-state levels of proteins involved in ATR ac- a marker of viral replication centers (21). At early times after tivation at appropriate times postinfection, using MRE11 degra- infection, TOPBP1 was recruited to viral replication sites (Fig. dation by both Ad5 and Ad12 as a control (Fig. 1A). DNA ligase IV S1); significantly, however, this staining pattern was lost at later (LIG4), previously shown to be targeted for degradation in Ad5- times (Fig. 2C), consistent with Western blotting data shown in infected cells (22), was also degraded in Ad12-infected cells, in- Fig. 1. Weak residual TOPBP1 nuclear staining was present at late dicating that, like MRN, it may be a common target for human times after infection, but this was not observed at viral replication Ads. Further analysis revealed that ATR, ATRIP, RPA70, RPA32, centers (Fig. 2C). These results indicate that, although TOPBP1 is and RAD9 levels remained constant in Ad5- and Ad12-infected initially recruited to viral replication sites, it is subsequently de- cells. Slower migrating forms of RAD9 and RPA32 were detected graded in Ad12-infected cells.