Infection with E1B-mutant adenovirus stabilizes p53 but ... - Nature

Oncogene (2011) 30, 865–875

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ORIGINAL ARTICLE

Infection with E1B-mutant adenovirus stabilizes p53 but blocks p53 acetylation and activity through E1A I Savelyeva1,2 and M Dobbelstein1,2 1

Department of Molecular Oncology, Go¨ttingen Center of Molecular Biosciences, Ernst Caspari Haus, University of Go¨ttingen, Go¨ttingen, Germany and 2Medical Biotechnology Center, Institute for Medical Biology, University of Southern Denmark, Odense C, Denmark

Wild-type adenovirus type 5 eliminates p53 through the E1B-55 kDa and E4-34 kDa gene products. Deletion or mutation of E1B-55 kDa has long been thought to confer p53-selective replication of oncolytic viruses. We show here that infection with E1B-defective adenovirus mutants induces massive accumulation of p53, without obvious defects in p53 localization, phosphorylation, conformation and oligomerization. Nonetheless, p53 completely failed to induce its target genes in this scenario, for example, p21/CDKN1A, Mdm2 and PUMA. Two regions of the E1A gene products independently contributed to the suppression of p21 transcription. Depending on the E1A conserved region 3, E1B-defective adenovirus impaired the ability of the transcription factor Sp1 to bind the p21 promoter. Moreover, the amino terminal region of E1A, binding the acetyl transferases p300 and CREB-binding protein, blocked p53 K382 acetylation in infected cells. Mutating either of these E1A regions, in addition to E1B, partially restored p21 mRNA levels. Our findings argue that adenovirus attenuates p53-mediated p21 induction, through at least two E1Bindependent mechanisms. Other virus species and cancer cells may employ analogous strategies to impair p53 activity. Oncogene (2011) 30, 865–875; doi:10.1038/onc.2010.461; published online 11 October 2010 Keywords: adenovirus; E1A; E1B; p53; Sp1; p300

Introduction The tumor suppressor p53 was initially identified and characterized by its interactions with viral oncoproteins. E1A and E1B represent the key oncogenes of adenovirus; E1A proteins bind the retinoblastoma family of proteins, whereas E1B-55 kDa forms a specific complex with p53. In cells infected with adenovirus type 5, p53 activity is antagonized by E1B-55 kDa and E4-34 kDa. E1B-55 kDa inhibits p53-mediated transcriptional activation. Together with E4-34 kDa, it also promotes ubiquitination and Correspondence: Professor M Dobbelstein, Department of Molecular Oncology, Go¨ttingen Center of Molecular Biosciences, Ernst Caspari Haus, University of Go¨ttingen, Justus von Liebig Weg 11, Go¨ttingen, Lower Saxony 37077, Germany. E-mail: [email protected] Received 24 June 2010; revised 12 August 2010; accepted 13 August 2010; published online 11 October 2010

proteasomal degradation of p53, presumably helping to avoid the premature death of infected cells (Levine, 2009). Many attempts were made to construct adenoviruses capable of replicating selectively in cancer but not in normal cells. Bischoff et al. (1996). hypothesized that E1B55 kDa-deleted viruses are not able to inactivate p53 and therefore, should replicate selectively in cells with mutated/ null p53 status, but not in cells bearing a wild-type p53 gene. This idea made E1B-deleted viruses a potentially attractive tool for cancer therapy. Some studies initially supported the idea of a E1B-55 kDa-deleted virus, ONYX-15, replicating in p53-deficient tumor cells (Heise et al., 1997). However, more recent reports addressed virus replication in a large variety of cell species with different p53 status; the observations suggested that infection with viruses lacking E1B-55 kDa results neither in selective replication nor in selective cell killing in the absence of functional p53 (Goodrum and Ornelles, 1998; Rothmann et al., 1998; Turnell et al., 1999). Importantly, we and others have previously observed impaired p53 function in adenovirusinfected cells, even when E1B-55 kDa was deleted (Ray et al., 1989; Hobom and Dobbelstein, 2004; O’Shea et al., 2004). Identifying the underlying reasons for continued p53 attenuation in this context may not only explain adenovirus-cell interactions, but may also point out general pathways of p53 regulation. In this study, we have investigated the molecular pathways of p53 attenuation by adenoviruses deleted for E1B-55 kDa. Despite p53 accumulation, p53-responsive genes were not induced. Surprisingly, the adenovirus E1A proteins prevented the activation of p21 through p53 by two distinct mechanisms: E1A blocked the acetylation of p53 at K382 and it reduced the amount of promoterbound Sp1, a transcription factor that cooperates with p53. Both the acetylations at K382 and transcription factor Sp1 are required for transcriptional activation of a number of p53 targets. Therefore, we propose that adenovirus may employ at least two distinct mechanisms of p53 inactivation that depend on E1A but not E1B. Results E1B-55 kDa mutant adenoviruses stabilize and accumulate inactive p53 To assess the influence of adenovirus infection on p53 levels and activity, A549 cells (containing wild-type p53)

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Infection with E1B-55 kDa-mutant adenoviruses does not detectably affect the localization, conformation, phosphorylation, or oligomerization of p53 To address whether E1B-55 kDa-mutant viruses are inactivating p53 by mislocalization, we detected p53 by immunofluorescence. p53 was found in the nucleus of cells infected with dl338 and R240A (Figure 2a); the staining was evenly distributed and did not merge with viral replication centers, as detected by antibodies to the E2A DNA binding protein. Thus, the lack of p53 activity cannot be attributed to improper localization in infected cells. For its function as a transcription factor, p53 requires an active conformation, which is disrupted by cancerassociated mutations. These structural mutants display epitopes that are normally hidden and one of these are reactive with the monoclonal antibody 240. To define whether E1B-55 kDa-deleted viruses accumulate p53 in an inactive or mutant-like conformation, immunoprecipitation analysis with antibodies, recognizing wild-type (1620) versus mutant (240) conformation of the protein, was performed. We thereby found that p53 retains wildtype conformation after infection (Figure 2b). Phosphorylations of p53 at Ser-15 (by DNA-dependent protein kinase and ataxia telangiectasia mutated/ ataxia telangiectasia-mutated and rad3-related) and Ser46 (by homeodomain-interacting protein kinase 2) contribute to p53 activity (Bode and Dong, 2004). We detected these modifications on p53 from infected cells by immunoblot analysis, to a degree comparable to camptothecin-treated cells (Figure 2c). p53 exists mostly as a tetramer and oligomerization of p53 is required for its function. We tested the ability of p53 to form oligomers after infection with E1B-55 kDamutant viruses, using glutaraldehyde cross linking, Oncogene

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were infected with adenovirus mutants bearing a deletion of E1B-55 kDa (dl338) or a point mutation (R240A) that disrupts its interaction with p53 (Shen et al., 2001). Wild-type adenovirus (dl309) was used as a control. p53 accumulated to high amounts 24 h after infection with dl338 or R240A (Figure 1a), but not with a replication-defective, E1-deleted virus vector (Supplementary Figure S1A). Wild-type virus suppressed p53 levels below detectability, in agreement with the essential role of E1B-55 kDa in p53 degradation (Figure 1a). However, the elevated levels of p53 in response to dl338- or R240A-infection did not increase the amount of its target gene products p21 and Mdm2; on the contrary, those were downregulated after infection (Figure 1a), as found earlier in similar experiments, even when the cells had been treated with camptothecin to trigger a DNA damage response (Hobom and Dobbelstein, 2004). To understand the mechanisms of p53 accumulation, we performed quantitative PCR analysis of p53 mRNA from infected cells, but did not detect changes comparable to the protein levels (Figure 1b). Instead, cycloheximide chase analyses indicated the stabilization of the p53 protein upon infection with E1B-55 kDa-deficient viruses (Figure 1c).

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Figure 1 Stabilization and accumulation of inactive p53 after infection with E1B-55 kDa mutant adenoviruses. A549 cells were mock-infected, infected with-wild type adenovirus (dl309) or with adenovirus mutants dl338 (lacking E1B-55 kDa) and R240A (carrying a substitution R–A at position 240 of E1B-55 kDa, resulting in strongly reduced p53 binding), at a multiplicity of infection (MOI) of 20. For a positive control, cells were treated with the DNA-damaging topoisomerase inhibitor camptothecin (CPT). Twenty-four hours after infection, the cells were harvested. (a) p53, p21 and Mdm2 proteins were detected by immunoblot analysis with the corresponding antibodies; b-actin and HSC70 were stained as loading controls. (b) Total RNA was isolated, reverse transcribed and subjected to real-time PCR analysis with primers amplifying p53 cDNA. Results were normalized to GAPDH mRNA. Mean values from three independent experiments are shown with standard deviations. (c) Twenty-four hours after infection, the cells were treated with the inhibitor of translation cycloheximide for the indicated time intervals, or with the dimethyl sulfoxide solvent alone for 4 h. p53 levels were determined by immunoblot analysis. Note that a longer exposure was used in the case of mock-infected cells.


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SDS–PAGE and immunoblot analysis. p53 from infected cells was compared with transiently expressed wild-type p53 or a mutant that lacks a functional oligomerization domain (p53DO; D327-347 (Atz et al., 2000)). As shown in Figure 2d, p53 from infected cells, as well as wild-type p53 but not p53DO, could be detected at molecular weights (around 55 and 130 kDa) that corresponded to monomeric and dimeric p53. This argues against the idea that the virus disturbs the oligomerization of p53. Taken together, all properties of p53 under study were compatible with its activity. Nonetheless, p53-mediated transcriptional activation is blocked by E1B-55 kDa mutant adenoviruses. E1A-deletion mutants differentially regulate p53-dependent gene expression Next, we assessed whether the E1A family of proteins is involved in the inactivation of p53 upon infection with E1B-55 kDa-deleted virus. The major products obtained by splicing of the primary E1A mRNA transcript are E1A-12S and 13S. These proteins differ only in a 46amino-acid domain, termed conserved region 3 (CR3), that belongs exclusively to E1A-13S. E1A was reported to affect p53 levels and activity, but this was mostly addressed upon transient overexpression of E1A through plasmid transfection (Lowe and Ruley, 1993; Steegenga et al., 1996). To understand the involvement of E1A proteins in p53 inactivation during virus infection, we compared a panel of Ad5 mutants carrying E1A mutations combined with a deletion or mutation of E1B (Figure 3a). These mutants also lack the antiapoptotic E1B-19 kDa protein. However, we observed that the additional deletion of E1B-19 kDa did not affect p21 expression in the context of a E1B-55 kDa-mutant virus (Supplementary Figure S1B). After infection of A549 cells, we analyzed the expression of two p53-activated genes, p21 and mdm2, by quantitative RT–PCR (Figure 3b, right). Interestingly, only viruses expressing E1A-13S reduced p21 and mdm2 mRNA levels more than 10-fold, whereas mutants bearing only E1A-12S (despite equal or greater overall E1A protein levels) failed to repress p53-responsive genes. Instead, E1A-12S-only viruses were upregulating p21 and mdm2 mRNA two- to sixfold, compared with mock-infected cells (Figure 3b, right). A similar pattern was observed in non-transformed cells (Supplementary Figure S2) and also for the transcription of PUMA, a proapoptotic target gene of p53 (Figure 3c). Of note, dl520-E1B–, a mutant lacking E1A-13S, was upregulating p21 mRNA, but not p21 protein levels (Figure 3b). In contrast, p21 protein levels were restored upon infection of cells with viruses that expressed E1A-mutants with deletions in the amino terminal portion or in the CR 2 (dl1101-E1B–, dl1107E1B– and dl0107-E1B–). This argues that adenovirus infection interferes with p21 translation and/or protein stability by unknown mechanisms that depend on additional E1A activities. In any case, our results show that E1A-13S, but not E1A-12S, blocks the accumulation

of p21 mRNA. p21 pre-mRNA levels were responding to the virus mutants much like the fully processed mRNA (Figure 3d), arguing that the regulation by E1A-13S occurs at the level of transcription, not RNA processing. Viruses with or without the deletion of E1A-13S differ in their ability to replicate (Carlock and Jones, 1981), and accordingly, virus replication centers were observed in a lower percentage of cells upon infection with dl338 versus dl520-E1B– (Supplementary Figure S3, A and B). However, this difference could be adjusted by treating the dl338-infected cells with an inhibitor of DNA synthesis, cytosine-arabinoside (AraC; Supplementary Figure S3C). Similarly, the two- to threefold difference between dl338 and dl520-E1B– with regard to early and late mRNA expression levels could be adjusted by AraC treatment (Supplementary Figure S4). Importantly, and despite the fact that AraC increases p21 mRNA levels in mock-infected cells, we still observed that p21 mRNA was suppressed upon infection by dl338 regardless of AraC treatment (Supplementary Figure S5). We conclude that the failure of dl520-E1B– to interfere with p21 mRNA accumulation is not due to impairments in virus replication. Furthermore, dl520-E1B– reached hexon levels similar to dl338 after 36 h after infection (Supplementary Figure S6A), but p21 mRNA levels remained far higher in dl338- than in dl520-E1B– infected cells for at least 48 h (Supplementary Figure S6B). To exclude a role for E4orf6 in differential p53 regulation (Dobner et al., 1996), we assessed p21 mRNA levels upon infection with a virus that carries E1A-13S, but l acks both E1B-55 kDa and E4orf6. In this case, p21 mRNA was still reduced compared with mock-infected cells (Supplementary Figure S7). Taken together, these results further support the idea that the CR3 domain of E1A suppresses p21 mRNA synthesis in adenovirusinfected cells. E1A-13S is responsible for removing Sp1 from the p21 promoter Despite accumulation of massive amounts of p53 (Figure 4a), both dl520-E1B– and dl338 only permitted low p53-DNA-association, comparable to the noninfected samples, as revealed by chromatin immunoprecipitation (Figure 4b). In contrast, camptothecin enriched p53 binding to the p21 promoter more than 10fold. We conclude that infection with E1B-55 kDadeleted adenovirus can generally decrease p53–DNA complex formation, but this does not explain the differential p53 target gene expression upon infection with dl520-E1B– versus dl338. The transcription of p21 and Mdm2 also depends on the transcription factor Sp1 acting synergistically with p53 (Lagger et al., 2003, Zhao et al., 2006). The E1A13S protein, in turn, was reported to bind Sp1 within its CR3 region (Liu and Green, 1994). Thus, we hypothesized that E1A-13S may impair the function of Sp1 in p21 transactivation. The viruses dl520-E1B– and dl338 enhanced the Sp1 protein levels to a similar extent (Figure 4a). However, chromatin immunoprecipitation of Sp1 retrieved significantly less p21 promoter DNA, Oncogene


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when the cells were infected with dl338, containing E1A-13S, compared with dl520-E1B–, bearing only E1A-12S (Figure 4c). Similarly, RNA polymerase II p35

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suggesting that the presence of E1A-13S reduces the formation of a functional transcription initiation complex. Finally, a pharmacological antagonist of the Sp1DNA-interaction, mithramycin A (Ray et al., 1989), suppressed p21 mRNA levels in dl520-E1B– infected cells (Figure 4d); this further argues that the maintenance of Sp1 activity is at least one reason why viruses lacking E1A-13S fail to suppress p21 mRNA synthesis. The amino terminal portion of E1A is required to abolish p53 acetylation at K382 Acetylation of p53 at lysine residues can activate transcription. Immunoblot analysis with antibodies recognizing p53 acetylated at position K382, after infection with dl338 or dl520-E1B–, showed that both viruses prevent the acetylation of p53 at this residue even in the presence of the DNA-damaging drug camptothecin (Figure 5a). Interestingly, however, infection with adenovirus mutant dl1101-E1B–, bearing an amino terminal mutation of E1A that prevents its binding to the histone acetyltransferase p300, allowed acetylation of p53 at K382 (Figure 5a). Another pair of virus recombinants, differing only with regard to the amino terminal region of E1A, showed the dependence of p53 acetylation on the mutation of E1A (Supplementary Figure S9). Thus, p300 binding by E1A represents a plausible mechanism for the missing p53 acetylation in infected cells. To understand whether acetylation of p53 at K382 is required for p53-mediated transactivation even in the presence of E1A-13S, we analyzed p21 mRNA levels after infection with a recombinant adenovirus that combines the mutations R2G in E1A (abolishing p300 binding) and R240A in E1B-55 kDa (deleting p53 binding). We had previously observed reduced p21 protein levels upon infection with this virus (Shen et al., 2001). However, this does not necessarily imply p21 mRNA suppression (cf. Figure 3b, dl520-E1B–). Remarkably, the E1A-R2G/E1B-R240A double mutant virus accumulated p53 with acetylated at K382 (Figure 5b) and allowed p21 transcription at least to the level of mock-infected cells, whereas the E1B-R240A single mutant suppressed p21 mRNA levels threefold (Figure 5c). This argues that the impairment of p53 acetylation by E1A represents another way by that the virus attenuates p53 activity, independently of E1ACR3 and E1B-55 kDa.

Discussion The presence of direct p53 antagonists in small DNA tumor viruses has led to the discovery of p53 in the first place. It therefore came as a surprise that adenoviruses lacking p53-binding E1B-55 kDa still abolish the efficient expression of p53-responsive genes, despite the accumulation of p53 in unusually high amounts. Our results largely explain this seeming contradiction. In addition to E1B-55 kDa, the E1A gene products also antagonize p53 during infection. Although E1A proteins do not form detectable complexes with p53, they avoid p53 activity at least in two ways. Firstly, the E1A CR3 region mediates p53 inhibition, and it interferes with Sp1-promoter-interactions. Secondly, E1A interferes with p53 acetylation through its amino terminal, p300-binding region. Both activities of E1A block the transcription of the major p53 target p21 even in the absence of a direct p53 antagonist (Figure 5d). It should be pointed out that this scenario reflects the situation of an infected cell, not oncogenic transformation by the virus E1 region. In the latter case, E1A induces apoptosis largely through p53 (Debbas and White, 1993). However, infected cells contain much higher amounts of virus proteins than transformed cells, with little time for the host cell to adapt the levels of cellular E1A-binding proteins. We propose that this scenario leads to p53-attenuation by E1A. Inactivation of p53 through E1A-13S The CR3 region of E1A turned out as a major determinant of how p53-responsive genes are transcribed upon adenovirus infection in the absence of E1B. Only virus mutants that retained the E1A-13S isoform—the sole isoform containing CR3—were still blocking the expression of such genes. Viruses without E1A-13S still interfered with p53 acetylation, but nonetheless allowed p53-induced gene expression. CR3 has long been known for its capability of interacting with the transcription factor Sp1 (Liu and Green, 1994), raising the possibility that E1A-13S antagonizes p53 by interfering with Sp1 activity. In agreement with this model, Sp1-DNA interaction was significantly diminished as a function of E1A-13S, and pharmacological inhibition of Sp1 reduced p21 levels when E1A-13S was absent (Figure 4). These observations do not exclude the

Figure 2 Post-translational modifications of p53 in cells infected with E1B-55 kDa-mutant adenoviruses. A549 cells were mockinfected or infected with the indicated adenovirus mutants at MOI of 5 (a) or 20 (b–d). (a) Twenty hours after infection or camptothecin addition, p53 and the viral E2A protein were detected by immunofluorescence and nuclei were visualized by 40 ,6diamidino-2-phenylindole. (b) Twenty-four hours after infection, the cells were harvested and subjected to immunoprecipitation (IP) with conformation-specific antibodies to p53: 1620 for wild-type p53, 240 for mutant p53. p53 levels were detected by immunoblot analysis (IB). C33A cells, bearing a p53 mutation recognized by the 240 antibody, p53 null H1299 cells, and the anti-T antigen antibody 419 served as controls. (c) Twenty-four hours after infection or camptothecin addition, the phosphorylation of p53 was analyzed by immunoblot, staining with phospho-specific antibodies to p53. (d) The oligomerization state of p53 was assessed by glutaraldehyde cross linking. Twenty-four hours after infection, the cells were harvested, treated with glutaraldehyde for 15 min at the indicated concentrations (%v/v), and subjected to SDS–PAGE. Cross-linked and monomeric p53 were then detected by immunoblot analysis with the antibody 1801 to p53. p53 from virus-infected cells was compared with p53 from H1299 cells transfected to express wild type p53 or the oligomerization mutant p53DO. Oncogene


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Figure 3 Differential regulation of p53-dependent gene expression by E1A- mutant viruses. (a) Table of viruses under study, containing short deletions within the E1A region and lacking the E1B-55 kDa and E1B-19 kDa coding regions, along with the capabilities of E1A mutants to interact with p300/CBP and Rb. Note that, although the deletion 111-123 has limited overlap with CR2 (121–139), this deletion still abolishes Rb binding (Shepherd et al., 1993). (b) A549 cells were mock-infected or infected with E1Amutant adenoviruses, described in A, at MOI 50. At the indicated time points after infection, the cells were harvested and subjected to immunoblotting (IB) and antibody staining of E1A, p53 and p21. Thirty-six hours after infection, Mdm2 and p21 mRNA levels were determined by reverse transcription and quantitative PCR (quantitative RT–PCR) and normalized to GAPDH mRNA. The ratio of mRNA levels relative to mock-infected cells is displayed in each case. (c) A549 cells were mock-infected or infected either with dl338 or dl520-E1B– at MOI 50. Twenty-seven hours after infection, total RNA was reverse transcribed and analyzed by quantitative RT–PCR. PUMA mRNA levels, normalized to GAPDH mRNA, were quantified as in b. (d) The cDNA samples from b were subjected to RT–PCR to quantify the pre-mRNA of p21, using primers to intron 1. In (b–d) mean values from three independent experiments are shown with standard deviations.

interaction of CR3 with other proteins as additional mechanistic bases for p53 inactivation. However, it is intriguing that Sp1 has previously been found as a critical determinant of p53 activity. Sp1 is required for efficient induction of p21 by p53 (Lagger et al., 2003; Zhao et al., 2006), and the same is true when p53 induces Mdm2 expression. A cancer-associated polyOncogene

morphism affects the interaction of Sp1 with the Mdm2 promoter, showing that this interaction is crucial for proper regulation of p53 activity (Bond et al., 2004). For activation of the proapoptotic targets PUMA and Bax, p53 also cooperates with Sp1 (Thornborrow and Manfredi, 2001; Koutsodontis et al., 2005; Wang et al., 2008).


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Figure 4 Association of Sp1 with the p21 promoter as a function of E1A-13S. A549 cells were mock infected, infected with dl338 or dl520-E1B– at MOI 50, or treated with camptothecin for 24 h. Chromatin immunoprecipitation (ChIP) was performed with the indicated antibodies, followed by quantitative PCR. (a) p53 and Sp1 were detected by immunoblot analysis. (b) p53 binding to its cognate p21 promoter element, located at -2283 upstream of the transcription start site, was quantified by ChIP, using antibodies to p53 or control antibodies (HA). Mean values from three independent experiments are shown with standard deviations. (c) Sp1 binding at the transcription start site of the p21 gene was detected in analogy to (b). Mean values from four independent experiments are shown with standard errors. P-value indicates the level of significance by that the precipitated amounts of promoter DNA are different, as determined by Student’s two-sample t-test. (d) A549 cells were infected as indicated for 24 h followed by treatment with the Sp1 inhibitor mithramycin A for 8 h. p21 mRNA levels were determined by quantitative RT–PCR as in Figure 3b. Mean values from three independent experiments are shown with standard deviations. The p21 mRNA levels upon infection with dl520-E1B– in the presence or absence of mithramycin A were different with a P-value of 0.032 (Student’s t-test). In contrast, the difference in p21 mRNA levels within mock-infected cells only showed a borderline significance (P ¼ 0.167) and may be due to experimental variations or to unknown, non-specific effects of the drug.

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Figure 5 Acetylation of p53 after infection with adenovirus mutants. (a) A549 cells were mock-infected, infected with dl338, dl520-E1B– or dl1101-E1B– at MOI 50, and/or treated with camptothecin. Twenty-four hours after infection, immunoblot analysis with antibodies to acetyl-p53 (K382) or global p53 was performed. (b) A549 cells were mock-infected or treated with camptothecin for 24 h, or infected with a recombinant adenovirus that carries the mutation R240A in E1B-55 kDa, in combination with the mutation R2G in E1A (resulting in strongly reduced p300 binding), at MOI 20 for 36 h. The cells were harvested and subjected to immunoblot detection of acetylated or global p53 as in a. (c) A549 cells were mock-infected or infected with the indicated virus mutants at MOI 20. Thirty-six hours after infection, p21 mRNA was quantified by RT–PCR and normalized to mtRNR2. Mean values from three independent experiments are shown with standard deviations. (d) Suggested model for regulation of p53 activity by adenovirus. Two distinct domains of E1A independently interfere with p53-mediated transcription, blocking p53 acetylation as well as the Sp1-DNA interaction. Note that the two mechanisms each contribute to the ability of adenovirus to attenuate the expression of p21/CDKN1A; their relative contribution (and that of other p53-regulatory mechanisms) may be different for other p53-responsive genes. In any case, however, E1A-13S is also required for the suppression of Mdm2 and PUMA in adenovirus-infected cells (cf. Figure 3).

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Blockage of p53 acetylation by E1A Adenovirus infection eliminated any detectable p53 acetylation at K382, the major p300-acetylation site (Avantaggiati et al., 1997; Lill et al., 1997; Ito et al., 2001). In contrast, when the amino terminal, p300-binding region of E1A (Harlow et al., 1986) was mutated in addition to E1Bdeletion, p53 not only accumulated in infected cells but also displayed strong K382 acetylation. It is thus conceivable that the interaction of the E1A-amino terminal region with p300, the related CREB-binding protein (CBP), and possibly P300/CBP-associated factor, contribute to the lack of p53 acetylation. This is further supported by structural analysis suggesting a competition of p53 and E1A for CBP binding (Ferreon et al., 2009) and by the ability of E1A to repress numerous genes with antivirus activity (Ferrari et al., 2008). Thus, the amino terminal region and the CR3 of E1A provide at least two independent but cooperating functions to antagonize p53 activity. Post-transcriptional regulation of p53 levels Adenovirus infection also affects the protein levels of p53 and p21 independently of their mRNAs. The halflife of p53 is strongly increased in response to infection with E1B-less viruses. We suspect that the attenuated expression of Mdm2 contributes to this phenomenon. In addition, adenovirus E1A proteins were shown to block proteasomal activity towards p53 by binding directly to proteasomal subunits (Turnell et al., 2000). This effect may be of particular importance in infected cells, as their E1A levels are higher than in E1-transformed cells. Multiple mechanisms for p53 inactivation Two well-described mechanisms contribute to inactivation of p53 during adenovirus infection: direct binding by E1B-55 kDa and ubiquitination mediated by E434 kDa. Our study suggests that two additional mechanisms attenuate p53 in infected cells, at least with regard to p21 induction. Firstly, transcription and Sp1-DNA association are impaired by E1A-CR3; secondly, p53 acetylation is abolished through the amino terminal, p300-binding region of E1A. Subsets of these mechanisms are still sufficient to prevent any detectable p53 activity. Perhaps, the parallel existence of these mechanisms provides an evolutionary advantage to the virus only in the context of infected tissue, with intact immune responses and associated proapoptotic mechanisms. Indirect mechanisms to inactivate p53 are prevalent in other viruses, too. Many non-oncogenic human papillomaviruses lack the ability to degrade p53 through their E6 gene products but still survived during evolution. In these cases, indirect p53 inactivation by interaction of E7 with p300 may prevent cell death (Bernat et al., 2003). Herpes virus family members do not directly antagonize p53 either, but at least in the case of cytomegalovirus (human herpes virus type 5), the virus protein IE2 binds p300 and thus attenuates p53 activity (Hsu et al., 2004). The tat protein of human immunodeficiency virus also binds p300 and PCAF and it attenuates p53 activity (Harrod et al., 2003; Wong et al., Oncogene

2005). Such indirect mechanisms may therefore suffice to avoid p53-induced cell death in many cases. A role of p53 for oncolytic virus selectivity? Adenoviruses lacking the interaction between E1B55 kDa and p53 have been examined for a long time as to their ability to replicate selectively in tumor cells with impaired p53 function, but with limited success. The prototype virus employed for this purpose (Bischoff et al., 1996) was later shown to display selectivity for some tumor cells, but not based on p53 activity. Rather, it takes advantage of differential mRNA export (O’Shea et al., 2005). The results presented here provide an explanation why the lack of E1B-55 kDa cannot be expected to result in p53-selective cytotoxicity. The question remains whether additional E1A mutations not only restore p53 activity but also allow virus replication preferentially in p53-mutant cells. In any case, however, it should be kept in mind that E1A mutations may reduce virus spread in tissues and hence result in a tumor-selective, but generally attenuated virus with limited therapeutic use. p53 inactivation in the absence of adenovirus infection Some of the mechanisms that directly attenuate p53 in adenovirus-infected cells may also abolish the tumorsuppressing function of p53 in cancer cells, especially when the cellular Mdm2 protein (representing the functional analog to E1B-55 kDa) is not sufficiently expressed. Some cancer cells delete or mutate p300 and/ or CBP, and CBP-mutations form the basis for a syndrome of cancer-proneness (Iyer et al., 2004), arguing that p300/CBP inactivation and a resulting lack in p53 acetylation may contributed to virus-independent cancer as well. Moreover, the MYC family of oncoproteins shows many functional analogies to E1A. MYC interacts with Sp1 and attenuates p21 expression (Gartel et al., 2001); moreover, it binds p300 (Faiola et al., 2005), perhaps modulating p53 acetylation. Finally, the inactivation of p53 by adenovirus infection is reminiscent of the Chk1-dependent failure of p53 to activate p21 expression during S-phase arrest (Gottifredi et al., 2001; Beckerman et al., 2009). The mechanistic principles used by adenoviruses to attenuate p53 activity in infected cells may thus serve as guidelines for the definition of p53-inhibitory pathways in human cancer. Materials and methods Cell culture and drug treatment A549, C33A and H1299 cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Immortalized keratinocytes, EPC-hTERT (Harada et al., 2003), were grown in serum free medium with supplements (0.2 ng/ml epidermal growth factor and 25 mg/ml bovine pituitary extract). Camptothecin was used at 300 nM, cycloheximide at 100 mg/ml, AraC at 50 mg/ml and mithramycin A at 1 mg/ml. Virus expansion and titration The viruses under study were: human Ad5 parental virus dl309 (Jones and Shenk, 1979), its derivatives dl338 (Pilder et al.,


873 1986) and R240A (Shen et al., 2001), bearing a deletion or a point mutation in the E1B-55 kDa coding region, respectively; E1B2xmut and E1AR2G, derived from R240A, carrying an additional deletion of the E1B-19 kDa coding region or the substitution R2G in E1A, respectively (Hobom and Dobbelstein, 2004); dl520-E1B– (encoding E1A-12S, but not E1A-13S and lacking the E1B region) and its E1A-mutant derivatives dl1101-E1B–, dl1107-E1B– and dl0107-E1B– (Shepherd et al., 1993) were kindly provided by J S Mymryk, R J A Grand and X. Zhang. E1A-13S-containing adenovirus mutants, bearing either a deletion of E1B-55 kDa (dl110) or a double deletion of E1B-55 kDa and E4-34 kDa (dl1017) (Bridge and Ketner, 1990) were obtained from G Ketner. An adenovirus vector, lacking the entire E1 region but expressing b-galactosidase was described (Koch et al., 2001). Viruses were grown in H1299 or HER911 cells and titrated in A549 cells, as described previously (Weigel and Dobbelstein, 2000). Virus stocks were stored in phosphate-buffered saline. Mock infections (control experiments) were performed by overlaying cells with phosphatebuffered saline alone, rather than with virus suspensions. Immunoblot analysis After SDS–PAGE and semidry transfer to nitrocellulose, the membranes were incubated with antibodies in phosphatebuffered saline containing 5% non-fat milk powder and 0.1% Tween 20. Primary antibodies used for detection were: antip53 (DO1, Santa Cruz Biotechnology, Santa Cruz, CA, USA; used for p53, unless indicated otherwise), anti-p53 (1801, Santa Cruz), anti-phospho-Ser15 p53 (16G8), anti-phosphoSer46 p53, anti-acetyl-Lys382 p53 (all Cell Signaling Technology, Danvers, MA, USA), anti-p21 (EA10, Calbiochem/ Merck, Darmstadt, Germany), anti-Mdm2 (2A10, hybridoma supernatant), anti-Sp1 (Upstate/Millipore, Billerica, MA, USA), anti-E1A (13S-5, Santa Cruz), anti-Hexon (HPR conjugated, Biogenesis Ltd., Poole, UK), anti-HSC70 (Santa Cruz) and anti-b-actin (Abcam, Cambridge, MA, USA). Detection was performed by HRP-conjugated antibodies (Jackson, West Grove, PA, USA) and chemiluminescence (Pierce/Thermo Scientific, Rockford, IL, USA). Quantitative RT–PCR RNA was isolated (Trizol, Invitrogen, Carlsbad, CA, USA), reverse transcribed using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA) and subjected to real-time PCR with the IQ SYBR Green Supermix (Bio-Rad). Primer sequences are provided in the Supplementary Table S1. Immunofluorescence Cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 and blocking with 10% fetal bovine serum, all in phosphate-buffered saline. Cells were co-stained with primary antibodies against p53 (rabbit FL-393, Santa Cruz) and adenovirus E2A protein (mouse B68, hybridoma supernatant), and secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes/Invitrogen, Carlsbad, CA, USA).

Immunoprecipitation with conformation-specific antibodies Cells were lysed in immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% NP40) and subjected to immunoprecipitation with 1 mg of antibodies (1620, 240, or 419, Calbiochem). Oligomerization studies Cells with wild-type or mutant p53 were harvested in lysis buffer (140 mM NaCl, 50 mM Tris HCl pH 7.6, 0.5% NP40, protein inhibitors; Roche, Basel, Switzerland), followed by cross linking with glutaraldehyde (0.0025%, 0.01%) for 15 min, and immunoblot analysis. The antibody 1801 (Santa Cruz) was used for p53 detection. Chromatin immunoprecipitation Chromatin immunoprecipitation was performed as described previously (Braun et al., 2008) with the antibodies against p53 (DO1, Santa Cruz), Sp1 (Upstate/Millipore), RNA polymerase II (N20, Santa Cruz, 1 mg) or the HA-tag (Santa Cruz; control). The precipitated DNA was analyzed by quantitative PCR with primers to the p21 promotor region (Supplementary Table S1).

Note added in proof While this paper was in press, another group reported that infection with an adenovirus lacking E1B-55 kDa results in the accumulation of inactive p53. Soria C, Estermann FE, Espantman KC, O’Shea CC. Heterochromatin silencing of p53 target genes by a small viral protein. Nature 466: 1076–1081.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements We thank JS Mymryk, RJA Grand, X Zhang and G Ketner for viruses, K Roemer for plasmids, AJ Levine for antibodies, OG Opitz for cells, C Hippel and A Dickmanns for excellent technical assistance, and S Johnsen for the help with ChIP experiments. The work was supported by the German Cancer Aid/Dr Mildred Scheel Stiftung, the EU 6th Framework Program (Integrated Project Active p53), the German Research Foundation (DFG), the Wilhelm Sander Stiftung, the Statens Sundhedsvidenskabelige Forskningsra˚d of Denmark, the Danish Cancer Society, and the Novonordisk fonden.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene