cellular and viral oncogenes , such as ras and adenovirus e1a , trigger p53 activation by inducing the expression of arf 9 , which inhibits mdm2 mediated p53 degradation .
arf is lost in 58% of cancers9 , which had previously been invoked as the critical factor that prevents p53 activation in e1b-55k infected tumor cells 23 . using a u2os stable cell - line ( p53 wild - type , arf negative ) in which arf expression is induced by isopropyl--d - thiogalactopyranoside ( iptg )
, we show that arf stabilizes p53 and activates p21 transcription in mock infection . nevertheless , although arf expression increases basal p53 activity , the induction of p21 is repressed in both wild - type ( wt ) and e1b-55k infected cells ( fig 1b and supp .
furthermore , endogenous arf induction also fails to activate p53 targets ine1b-55k infected saecs ( fig 1a ) .
thus , p53 is inactivated , irrespective of e1b-55k and arf expression in adenovirus infected cells .
dna damage signals also play a critical role in activating p53 , triggering p53 phosphorylation and protein stabilization 8,24 . in clinical trials , e1b-55k ( onyx-015 ) ,
was used in combination with genotoxic chemotherapies , such as 5-fluorouracil ( 5-fu ) 19,20 .
we reasoned that the induction of p53 levels alone may not be sufficient to activate p53 in infected cells , and that dna damage is also required .
therefore , we also analyzed e1b-55k infected saecs and show that p53 transcriptional targets can not be activated by  irradiation ( fig 1c and supp .
fig 6 ) , uv irradiation ( supp . fig 7 ) or doxorubicin ( dox , fig 1e ) .
the activation of p53 in response to dna damage is mediated via kinases , such as atm , atr , dna - pkcs , chk1 and chk2 , which phosphorylate p53 8 at key residues , stabilizing p53 and potentiating p53 dna binding 24 .
a possible explanation for the failure of dna damage to activate high p53 levels in e1b-55k infected cells is that p53 phosphorylation is inhibited by viral infection .
however , even without the introduction of exogenous genotoxic stress , p53 is already highly phosphorylated at multiple sites targeted by dna damage kinases in e1b-55k infected saecs ( fig 1d ) . thus , although oncogenes and dna damage trigger p53 stabilization and phosphorylation ine1b-55k infected cells , p53 fails to activate the transcription of downstream effectors .
we next examined if in the absence of e1b-55k , mdm2 binds and inactivates p53 in adenovirus infected cells .
in contrast to mock , nutlin fails to stabilize p53 further or induce p21 in e1b-55k infected saecs ( fig 1e ) .
the histone deacetylase ( hdac ) inhibitor , trichostatin a ( tsa ) , induces the expression of p21 independently of p53 stabilization or phosphorylation ( fig 1e ) .
we conclude that p53 transcriptional targets are dominantly suppressed in adenovirus infected cells , irrespective of e1b-55k , and can not be activated in response to radiation , genotoxic drugs , arf , mdm2 antagonists or hdac inhibitors .
our data strongly suggest that there is a previously undiscovered adenoviral protein that inactivates p53 independently of p53 degradation . to test this
, we screened for p53 activation in primary cells infected with adenoviruses that have compound mutations in e1b-55k and other early viral genes ( supp .
in addition to deleting e1b-55k , the loss of either e1a-13s or e4-orf3 is required to activate p53 in infected cells ( fig 2a ) .
this is surprising , especially since e1a is a potent oncogene that triggers p53 activation in cellular transformation 7,14 . in adenovirus infection
, the e1a-13s splice form is required for the transactivation of other viral genes 17 , including e4-orf3 ( fig 2a ) .
consistent with this , we show that in contrast to gfp , the ectopic expression of e4-orf3 rescues p53 inactivation in both e1b-55k/e4-orf3 and e1b-55k/e1a-13s infected cells ( fig 2b ) .
the slight reduction of p21 by ad - gfp ine1b-55k/e4-orf3 co - infection is due to the partial activation of e4-orf3 transcription ( in trans ) by e1a-13s , which does not occur ine1b-55k/e1a-13s co - infection ( supp .
hence , e1a-13s induces the expression of e4-orf3 which then inactivates p53 via an e1b-55k independent mechanism .
moreover , the expression of e4-orf3 alone is also sufficient to inhibit p53 activation ( supp .
these data reveal e4-orf3 as a novel adenoviral protein that inactivates the p53 tumor suppressor pathway .
the proposed p53 tumor selectivity of the e1b-55k oncolytic therapy , onyx-015 , is based on p53 stabilization being the sole critical event that determines p53 activated transcription .
although there is some basal p53 activity in e1b-55k infected cells compared to wild - type virus , the additional deletion of e4-orf3 is necessary for p53 to activate downstream effectors over the course of infection ( fig 2c - d ) .
in contrast to p53 transcriptional targets , the mrna levels of p53 and the housekeeping gene , gusb , are not impacted by e4-orf3 ( supp .
p53 stabilization is required to activate p53 transcriptional targets , and does not occur in e4-orf3 infection where p53 is degraded by e1b-55k / e4-orf6 .
fig 12 ) , we show that the induction of p21 and mdm2 in e1b-55k/e4-orf3 infection is p53-dependent . thus , the deletion of both e1b-55k and e4-orf3 is necessary to activate p53 in adenovirus infection .
we conclude that e4-orf3 has a critical and novel role in inactivating p53 independently of e1b-55k and p53 degradation .
dna tumor virus proteins , such as e1b-55k , sv40 lt and hpv e6 inactivate p53 via direct high affinity protein - protein interactions 1 . however , contrary to this established paradigm , e4-orf3 does not co - localize with p53 ( supp .
the induction of p53 levels and phosphorylation induces p53 conformational changes that drive sequence - specific dna binding and the recruitment of transcription co - factors 25 .
a p53 dna binding domain that is competent to bind to dna can be distinguished by immunoprecipitation with pab 1620 versus pab 240 26 .
p53 is immunoprecipitated selectively by pab 1620 in bothe1b-55k and e1b-55k/e4-orf3 infected cells ( supp .
fig 14 ) , demonstrating that the p53 dna binding domain is in a protein conformation that should be capable of binding to dna 26 in both cases . to functionally determine if e4-orf3 prevents p53 dna binding , we transfected u2os cells with a p53 luciferase plasmid ( p53-luc ) , where p53 binding to consensus dna sequences activates luciferase transcription 27
. a control pgl3-luciferase reporter ( non - p53 promoter ) is activated to similar levels in all viral infections ( supp .
infected cells , p53 activated transcription of luciferase is inhibited after 24 hours ( fig 3a ) , which is expected due to p53 degradation . in contrast , p53-luciferase is activated in both e1b-55k and e1b-55k/e4-orf3 infection ( fig 3a ) .
the induction of luciferase requires p53 dna binding , since a mutated p53 response element ( p53-mutant ) abolishes luciferase activity .
these experiments demonstrate that e4-orf3 does not compete with p53 for binding to consensus dna target sequences or prevent p53 transcriptional activation of promoters in ectopic reporter plasmids .
the ability of e4-orf3 to prevent p53 activated transcription of endogenous targets but not ectopic p53-luciferase plasmids is at first difficult to reconcile .
plasmid dna is not subject to the same architectural and packing constraints as dna in cellular chromatin .
therefore , we performed p53 chromatin immunoprecipitations ( chips ) to determine if e4-orf3 specifically prevents p53 dna binding in the context of cellular chromatin .
p53 binding to target sites in the p21 ( 5 and 3 site ) and mdm2 promoters 25 is induced upon doxorubicin treatment and e1b-55k/e4-orf3 infection , where it activates the transcription of p21 and mdm2 rnas ( fig 3b - c ) .
in contrast , although p53 is induced to similar levels , e4-orf3 prevents p53 dna binding to the p21 and mdm2 promoters in e1b-55k infected cells ( fig 3b  c and supp .
thus , e4-orf3 inactivates p53 by preventing p53 binding to dna target sites specifically in the context of cellular chromatin .
we reasoned that p53 dna binding depends not only on the protein conformation of p53 but also the accessibility of target promoters in the cellular genome .
we hypothesized that e4-orf3 could inactivate p53 by inducing heterochromatin at endogenous target promoters , preventing the access of p53 to dna .
heterochromatin compaction is specified by the loss of histone acetylation and induction of repressive histone methylation 28 .
tsa fails to induce p21 in e1b-55k infected saecs ( fig 1e ) suggesting that e4-orf3 inactivates p53 targets via a mechanism that is dominant to the inhibition of histone deacetylation . in cancer ,
the aberrant epigenetic silencing of tumor suppressor genes , such as p16 , is initiated by the methylation of histone h3 at lysine 9 ( h3k9 ) 29 .
however , in e1b-55k infected cells , where p53 is inactive , dense regions of h3k9me3 repressive heterochromatin are induced at the periphery of the nucleus ( fig 3d and supp .
, we show that suv39h1 and suv39h2 ( which share 59% sequence identity and have redundant functions ) 30,31 , but not setdb1 32 or g9a 33 , are specifically associated with the formation of de novo h3k9me3 heterochromatin domains in e1b-55k infected nuclei ( fig 3e ) .
the formation of these domains requires e4-orf3 and does not occur in either mock or e1b-55k/e4-orf3 infected cells ( supp .
these data demonstrate that e4-orf3 induces novel h3k9me3 heterochromatin , which could deny p53 access to endogenous target promoters . to test this , we performed p53 and h3k9me3 chips .
the induction of repressive heterochromatin by e4-orf3 is not associated with a global upregulation of either total histone h3 or h3k9me3 , which are at similar levels in all infections ( fig 4a ) . in e1b-55k/e4-orf3
infected cells , p53 binding is induced at p21 and mdm2 promoter sites ( consistent with fig 3c ) while h3k9me3 is at a similar level to an igg negative control ( fig 4a and supp .
in contrast , in e1b-55k infected cells , h3k9me3 is enriched at the p21 and mdm2 promoters where p53 binding is prevented .
h3k9me3 is also induced at the -5 kb region of the p21 promoter and is not restricted to p53 binding sites ( supp .
thus , in cells expressing e4-orf3 , there is an inverse correlation between p53 and h3k9me3 at p53 regulated promoters .
the same conclusions were reached for additional p53 targets , including gadd45a , fas , puma and pig3 ( supp .
in contrast , at non - p53 regulated promoters , such as actin and polr2 , h3k9me3 is not induced in e1b-55k infected cells relative to mock ( supp .
basal h3k9me3 is decreased at these promoters ine1b-55k/e4-orf3 infected cells , suggesting that e4-orf3 may also restrain global demethylase activity .
we conclude that e4-orf3 inactivates p53 by inducing de novo h3k9me3 heterochromatin silencing at p53 target promoters . with access denied
thus , a major question is how is e4-orf3 directly involved in inducing repressive h3k9me3 heterochromatin at p53 target promoters ?
e4-orf3 does not co - localize with p53 and forms a distinctive web - like structure in the nucleus ( supp .
fig 13 ) . we show that e4-orf3 demarcates the formation of de novo h3k9me3 heterochromatin domains ine1b-55k infected cells .
e4-orf3 is , for the most part , adjacent to h3k9me3 , suggesting it acts as a novel platform that catalyses heterochromatin formation through transient or long - range interactions ( supp .
, we show that e4-orf3 forms a continuous scaffold that specifies de novo heterochromatin assembly as it weaves through the nucleus ( fig 4b ) .
these data demonstrate a direct role for e4-orf3 in orchestrating h3k9me3 heterochromatin silencing at p53 target promoters .
furthermore , they reveal an extraordinary nuclear scaffold that either builds on existing architectural features that organize cellular dna or is a novel viral construction that targets heterochromatin assembly at p53 target promoters .
these data beg the question as to the specificity of e4-orf3 in silencing p53 targets . to determine the global consequences on cellular transcription , we performed genome - wide expression analyses on infected saecs ( supp .
these studies demonstrate that e4-orf3 is an exclusive player in the global transcriptional changes induced upon viral infection .
there are 1,730 overlapping genes that are similarly up or downregulated by a log fold change ( fc ) greater than two in bothe1b-55k and e1b-55k/e4-orf3 versus mock , which reflect a common transcriptional program ( fig 5a ) .
these global changes are associated with the cell cycle and e2f activation ( supplementary table i and ii ) .
this is consistent with e1a mediated inactivation of rb 34 and recruitment of p300 and pcaf to induce active histone acetylation marks at the promoters of genes involved in cell growth , division and dna synthesis 35,36 .
thus , e4-orf3 induced heterochromatin silencing , as well as the scaffold it forms throughout the nucleus , does not affect the global activation of cellular transcripts induced by viral infection . to define the genes specifically targeted by e4-orf3
e4-orf3 prevents the transcriptional activation of 265 genes by a log fold change of two or more in e1b-55k infected cells . to determine how many of these genes are likely to be regulated by p53 , we used two criteria : the presence of consensus p53 dna binding sites in their promoters and their induction upon treatment with the mdm2 antagonist , nutlin .
a heat map of top transcripts differentially upregulated in response to e1b-55k/e4-orf3 and nutlin includes well known p53 targets ( mdm2 , fas , pig3 , tp53inp1 , btg2 , lrdd ) associated with growth inhibition and apoptosis , as well as novel targets ( hrh1 , rnase7 , jmjd1c ) ( fig 5b and supp .
73% are induced in response to nutlin and/or have predicted p53 binding sites ( fig 5c and supplementary table iii 
a pathway analysis of e4-orf3 regulated transcripts indicates that in addition to the p53 pathway , there is a significant overrepresentation of genes associated with immune modulation as well as tissue / vascular remodeling ( supplementary table v ) .
these data suggest that e4-orf3 may target p53 promoters as part of a general anti - viral transcriptional silencing program , which is consistent with the highly defective replication of e1b-55k/e4-orf3 in primary cells ( supp .
the conclusions of our study challenge the general assumption that p53 induction and phosphorylation is tantamount to p53 activity , which is the premise for several cancer therapies 1113 .
our data reveal a novel and dominant mechanism of p53 inactivation that acts via the targeted epigenetic silencing of p53 target promoters .
we identify a viral protein , e4-orf3 , which appears to form a novel scaffold that weaves through the nucleus , directing suv39h1/2 h3k9me3 heterochromatin assembly at p53 target promoters to silence p53 activated transcription in response to genotoxic and oncogenic stress ( fig 5d ) .
remarkably , this suppressive nuclear web selectively ensnares p53 and anti - viral genes while operating in the backdrop of global transcriptional changes that drive pathological cellular and viral replication .
thus , a major question is if e4-orf3 reflects or exhorts an existing cellular mechanism and nuclear structure that censors p53 transcriptional activity .
strikingly , all of the known targets of e4-orf3 , pml 38 , the mre11/rad50/nbs1 ( mrn ) dna damage / repair complex 39 and tif1 40 are subverted by tumor mutations .
it is intriguing to speculate that e4-orf3 physically integrates the inhibitory effects of several cancer pathway mutations , both known and yet to be discovered , which together have emergent functions 4 in silencing p53 activity .
similar to the discovery of p53 with a viral protein 2,3 , e4-orf3 provides a powerful dynamic probe with which to define critical cellular factors that induce de novo epigenetic silencing of p53 target promoters in somatic cells .
this has important implications for understanding how high levels of wild - type p53 might also be inactivated in cancer as well as the dynamic mechanisms that induce aberrant epigenetic silencing of tumor suppressor gene loci .
finally , our identification of e4-orf3 changes the fundamental definition of how p53 is inactivated in adenovirus infected cells , which is a critical mechanistic insight that could now enable the rational development of true p53 tumor selective adenoviral therapies .
real - time quantitative pcr ( rt - qpcr ) was used to quantify p53 targets 21 , and normalized relative to 18s . for luciferase assays ,
100 um d - luciferin was added 4 hours post infection ( h.p.i . ) and luminescence quantified every hour .
global gene expression was determined using affymetrix human exon 1.0st arrays and analyzed with partek and genomatix software .
 the transcription factor p53 guards against tumor and virus replication and is inactivated in almost all cancers . 
 p53 activated transcription of target genes is thought to be synonymous with the stabilization of p53 in response to oncogenes and dna damage . during adenovirus replication , 
 the degradation of p53 by e1b-55k is considered essential for p53 inactivation , and is the basis for p53 selective viral cancer therapies . here 
 we reveal a dominant epigenetic mechanism that silences p53-activated transcription , irrespective of p53 phosphorylation and stabilization . 
 we show that another adenoviral protein , e4-orf3 , inactivates p53 independently of e1b-55k by forming a nuclear structure that induces de novo h3k9me3 heterochromatin formation at p53 target promoters , preventing p53 dna - binding . 
 this suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication . 
 these findings are important for understanding how high levels of wild - type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumor suppressor loci . 
 our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53 selective oncolytic viral therapies . 
p53 stabilization without activity
E4-ORF3 inactivates p53 independently of E1B-55k
E4-ORF3 prevents p53-DNA binding at chromatin
Repressive histone methylation silences p53 targets
Selective silencing of the p53 transcription program
Discussion and perspective
Methods Summary
Supplementary Material