431_ftp

Rev. Med. Virol. 2004; 14: 301319.Published online in Wiley InterScience (www.interscience.wiley.com).

Reviews in Medical Virology DOI: 10.1002/rmv.431

Human tumor suppressor p53 and DNA virusesS. Collot-Teixeira, J. Bass, F. Denis and S. Ranger-Rogez*Department of Virology, Limoges University Teaching Hospital, CHRU Dupuytren, 2 avenue Martin LutherKing, 87042 Limoges, France

SUMMARY

Human tumor suppressor protein p53 plays a major role in the cell cycle, orchestrating a number of important genesinvolved in cell-cycle control and apoptosis, and seems to be one of the most important molecules protecting cellsfrom malignant transformation. Mutations in the p53 gene are observed in about 50% of primary tumors, inducingdefective p53 protein no longer capable of binding DNA and of activating transcription. Certain DNA viruses arethought to act in a similar way and may also contribute to the progression of invasive cancer in infected tissue. One ofthe most effective strategies employed by these viruses is the inhibition of p53 protein by interaction with viraloncoproteins, implying a direct but also an indirect role of these viruses in the impairment of p53 structure andfunction. This article provides a summary of current knowledge concerning p53 tumor suppressor protein andreviews the different mechanisms adopted by different DNA viruses in undermining p53 function. Copyright # 2004John Wiley & Sons, Ltd.

Accepted: 23 February 2004

INTRODUCTIONHuman tumor suppressor protein p53 plays amajor role in the cell cycle. It is often describedas the guardian of the genome [1] because of itskey role in cell cycle arrest and in inducing apop-tosis when DNA becomes extensively damaged.

P53 gene mutations, catalogued in the Interna-tional Association of Cancer Registries (IARC)database (www.iacr.fr), are an extremely frequentfeature of many naturally occurring tumors. Theyare observed in about 50% of primary tumors [2],and in up to 85% of breast, lung and colon cancers.The mutated p53 allele encodes defective protein

that is no longer capable of binding DNA and ofactivating transcription. Certain viruses, impli-cated in human cancers, could participate in cellneoplasia by encoding viral proteins able to inhibittumor suppressor protein p53 activity (Table 1). Itimplies a direct but also an indirect role of theseviruses in the impairment of p53 structure andfunction.

This article summarises the different roles of p53in normal and stressed cells, its interactions,activations and inhibitions, in order to betterunderstand the implications of the differentmechanisms used by DNA viruses in abrogatingp53 functions in cell transformation.

P53

Protein descriptionHuman p53 is a phosphoprotein of Mr 53000encoded by a 20 kb gene containing 11 exons [3]and located on chromosome 17q13 [4]. This genebelongs to a highly conserved gene family contain-ing at least two other members; p51/p63 [5,6] andp73 [7]. The proteins share many properties andtheir intricate interplay serves to optimise and reg-ulate cell proliferation. P53 protein contains 393amino acids and is composed of several structuraland functional domains: an amino-terminaldomain required for transcription activation, aproline-rich sequence containing multiple copies

RR EE V II E W

Copyright # 2004 John Wiley & Sons, Ltd.

*Corresponding author: Dr S. Ranger-Rogez, Department of Virol-ogy, Unit EA3175, CHRU Dupuytren, 2 avenue Martin LutherKing, 87042 Limoges, France. E-mail: [email protected]

Abbreviations usedAkt, phosphatidylinositol 3-OH-kinase; ARF, ADP-ribosylation fac-tor 1; BRCA-1, breast cancer 1 gene; CAK, CDK-activating kinase;CBP, CREB binding protein; CDK, cyclin-dependent kinases;CDKI, CDK inhibitor; DP, dimerisation partner of E2F; E2F, E2 pro-moter binding factor; GADD45, growth arrest and DNA damageinducible gene; HBc, hepatitis B viral core protein; HBx, hepatitis Bviral X protein; HDM2, human double minute 2; HIF-1, hypoxia-inducible factor 1 alpha; ING, inhibitor of growth family; NES,nuclear export signal; NQO1, NADPH quinone oxidoreductase 1;PARP-1, poly(ADP-ribose) polymerase-1; PCAF, p300/CREB-binding protein associated factor; PCNA, proliferating cell nuclearantigen; PRb, retinoblastoma protein; RGC, ribosomal gene cluster;SV40, simian virus 40; TAFs, TBP-associated factors; TBP, TATA-binding protein; TRRAP, transformation/transcription domain-associated protein; XPC, xeroderma pigmentosum group C

of the PXXP sequence (where X is any amino acid),a central core DNA-binding domain, a flexiblelinker region, a tetramerisation domain and astrongly basic carboxyl-terminal regulatorydomain [8,9] (Figure 1). Most p53 mutations iden-tified in tumors occur at sites located on the centralcore DNA-binding domain [10]. The binding ofp53 to specific DNA could cause significant shiftsof residues on the DNA-binding interface thattranslated into the beta-sheet protein, and bindingof non-specific DNA, causes weak but qualita-tively similar shifts, corresponding to weakerbinding interactions [11].

Native state p53 protein contains large unstruc-tured regions in its N- and C-terminal parts. It is amodular protein consisting of defined structuredand unstructured regions. Lack of rigid structurecombined with low overall stability has been pro-posed as an explanation for its physiological inter-action with a multitude of associated proteins andthe regulation of its turnover [12].

General presentationUnder normal circumstances, p53 is a short-livedprotein maintained at very low or undetectablelevels bound to its major inhibitor HDM2 (humandouble minute 2). In response to damaged DNA,nucleotide depletion, hypoxia, oncogene expres-sion, metabolic changes or viral infections, p53accumulates in the nucleus and is activated as atranscription factor [13]. It then stimulates the

expression of genes implicated in cell cycle arrest,DNA repair or in apoptosis (Table 2).

Differential transactivation by the p53 transcrip-tion factor is highly dependent on p53 level andpromoter target sequence [14]. In response todamaged DNA, p53 protein undergoes extensivepost-translational modifications of at least 18 sitesalong its length [15] as a result of an activationprocess involving phosphorylations and acetyla-tions of the protein. During this activation process,six serines and two threonines in the N-terminaldomain are phosphorylated (Figure 1). Onehypothesis postulates that phosphorylations ofresidues on the p53 protein transform its tightlyfolded transactivation domain into a more openconformation that can interact with transcriptionfactors, thus enhancing gene expression [16]. Acet-ylations of p53 C-terminal domain, by PCAF(p300/CREB-binding protein associated factor)and by CBP/p300 [17,18] (Figure 1), are also neces-sary for the activation of the protein. Acetylationscould dramatically stimulate the sequence-specificDNA-binding activity of p53, possibly as a directresult of conformational change [18].

Activation of p53 occurs in three stages (a) anincrease in p53 protein concentration either byenhanced translation or increased half-life; (b)transformation of the p53 protein from latent toactive conformation; and (c) translocation of p53protein from the cytoplasm to the nucleus. P53protein has been found physically associated

Table 1. Viruses with malignant potential. List of viral proteins associated with p53 and pRb

Virus Human associated cancer p53 mutations p53 binding pRb binding

Adenovirus * * E1B55K/E4orf6 E1ASV40 * * Large T Antigen (LTag) LTagBKV/JCV Brain tumors * LTag LTagCMV Colorectal cancer * IE286 IE286EBV Burkitts lymphoma Frequent BZLF1/EBNA-5 EBNA-5HHV-6 Lymphoproliferative disorders Rare DR7 ?HHV-8 Kaposi sarcoma, Primary Infrequent LANA/v IRF/K8/ORF50 ?

effusion lymphoma,Multicentric Castlemansdisease

HSV-1 Oropharyngeal cancer (?) * ICP0 ?HBV Hepatocellular carcinoma Occasional HBx/HBc ?HPV (16/18) Cervical carcinoma Infrequent E6 E7

*Not described.

302302 S. Collot-TeixeiraS. Collot-Teixeira et al.et al.

Copyright # 2004 John Wiley & Sons, Ltd. Rev. Med. Virol. 2004; 14: 301319.

with tubulin in cellular microtubules, which whenactive, participate with dynein motor protein totransport p53 into the nucleus after damage toDNA [19].

The active form of p53 protein is tetrameric incomposition [20] (Figure 1). Biogenesis of p53involves co-translational dimerisation of mono-mers followed by post-translational dimerisationof the resulting dimers [21]. Once in its activatedtetrameric form, p53 protein binds with highaffinity to DNA and activates transcription frompromoters with consensus binding site 50-PuPuPu-C(A/T)(T/A)GpyPyPy-30 [22]. Activated p53 also

binds, via its amino-terminal activation domain,to TAFII31 (TBP associated factor) [23] and TAFII70proteins (co-activators of the p53 protein) [24]which are sub-units of TFIID. The latter is a multi-protein complex comprising the TATA-bindingprotein TBP and approximately ten associated fac-tors (TAFs) which form a key component of theRNA polymerase II transcription machinery [25].

Activated p53 protein functions principally as atrigger of gene transcription, controlling genesimplicated in cell cycle control, in DNA repair orin apoptosis (Table 2), as explained above. How-ever, it can also act as a negative regulator of geneswhose promoters lack p53-binding sites. Themechanism by which p53 exerts this inhibition isprobably in part through its physical interactionwith basal components of the transcriptionalmachinery such as TBP [26]. However, p53TBPinteraction alone is insufficient to induce repressionof transcription and the mechanism almost cer-tainly involves interactions between p53 and otherfactors such as TAFs that are also normallyrequired to activate transcription [27]. P53 repressesthe activity of promoters that depend on the pre-sence of a TATA-box. Promoters, whose accuratetranscription is directed by a pyrimidine-rich initia-tor element, are immune to the effects of p53 [28].P53 represses transcription of several genes suchas the IL-6 gene [29] and the pRb gene [30]. In addi-tion, it is also known to repress transcriptionmediated by RNA polymerase I [31], RNA poly-merase II [32] and RNA polymerase III [3335].

P53 inhibition of gene expression can also takeplace after transcription during the translationphase of mRNA. Data presented by Horton et al.[36] demonstrated that p53 causes a rapid decreasein initiation of translation.

Role of p53 in cell cycle arrest at theG1/S checkpointThe main mechanism employed by p53 to blockthe cell cycle at the G1/S checkpoint is basedon the inhibition of E2F proteins (E2 promoter bind-ing factor), which are transcription factors impli-cated in activation of genes required for G1/Stransition (Figure 2). E2F consists of heterodimerscontaining a sub-unit encoded by the E2F genefamily and a sub-unit encoded by the DP family.Under normal circumstances, E2F transcriptionfactors are blocked by binding to pRb protein orits relatives, p130 and p107 [37,38] thus inhibiting

Figure 1. Structure of p53 protein. (A) Domains of p53 with bind-

ing sites of cellular (below the diagram) and viral (above the dia-

gram) p53 partners. TAD, transcription activation domain; RPR,

rich proline region; DBD, DNA binding domain; FLR, flexible

linker region; TD, tetramerisation domain; RD, regulation

domain. (B) Structure of p53 monomer and tetramer. (C) Structure

of the C-terminal domain of p53 tetramer as determined by x-raycrystallography. Derived from Clore et al. Science, 1994 and Jef-frey et al. Science, 1995 and modified. The alpha helix of eachmonomer is shown in dark purple, dark blue, orange and dark

green. P, phosphorylation; Ac, acetylation

Human tumor suppressor p53Human tumor suppressor p53 303303


E2F transcription activity. At the end of the G1phase, pRb is phosphorylated by several cyclin/CDKs (cyclin dependent kinases) inducing achange in pRb conformation and the release ofan active E2F transcription factor. Free E2F acti-vates genes implicated in DNA synthesis drivingprogression of the cell cycle from G1 to S phases,but the exact mechanism of E2F-mediated activa-

tion of transcription remains unclear. E2Fs alsoregulate the expression of genes involved in differ-entiation, development, proliferation and apopto-sis [39].

In normal cells, p53 is maintained at a very lowlevel, coupled with HDM2, and exerts no effect.But in response to damaged DNA, a rapid increasein the amount of the active form of p53 protein

Table 2. List of p53 transcriptionally targeted genes

Genes activacted by p53 Function Authors

p21WAF1/Cip1 CDK inhibitor El Deiry et al., 1993Cell cycle regulation Harper et al., 1993

GADD45 DNA repair Carrier et al., 199414-3-3 DNA repair Hermecking et al., 1994MDM2 Inhibition of p53 Wu et al., 1993IRF-5 Immune response Mori et al., 2002Cyclin K Cell cycle regulation Mori et al., 2002Cyclin G Cell cycle regulation Okamoto et al., 1994B99/Gtse-1 Cell cycle regulation Collavin et al., 2000MUC2 Defence in lumen Ookawa et al., 2002ICAM-1 Cellcell interaction Gorgoulis et al., 2003ATF3 Response to stress cells Zhang, Gao et al., 2002MC610 Apoptosis Zhu et al., 2000Bax Apoptosis Miyashita et al., 1995IGF-BP3 Apoptosis Buckbinder et al., 1995PIG3 Apoptosis Polyak et al., 1997CD95/FAS Apoptosis Owen-schaub et al., 1995p85 Apoptosis Yin et al., 1998KILLER/DR5 Apoptosis Wu et al., 1997Apaf-1 Apoptosis Rozenfeld-Granot et al., 2002Zac-1 Apoptosis Rozenfeld-Granot et al., 2002mRTVP-1 (mouse gene) Apoptosis Ren et al., 2002PRG3 Apoptosis Ohiro et al., 2002PAG 608 Apoptosis Israeli et al., 1997Caspase-1 Apoptosis Gupta et al., 2001Caspase-6 Apoptosis MacLachlan et al., 2002PERP Apoptosis Attardi et al., 2000p53DINP1 Apoptosis Okamura et al., 2001Pidd (mouse) Apoptosis Lin et al., 2000PUMA Apoptosis Nakano et al., 2001Noxa ? Apoptosis Oda et al., 2000Scotin Apoptosis Bourdon et al., 2002PRG3 Apoptosis Ohiro et al., 2002PAC1 Apoptosis Yin et al., 2003TSAP6 Apoptosis Passer et al., 2003



stimulates transcription of genes like p21WAF1/CIP1

(Table 2) whose product is a CDKI (CDK inhibitor)[40]. The N-terminal domain of p21 interacts withand inhibits CDK-cyclin [41] halting phosphoryla-tion of pRb protein so that E2F remains inacti-vated, bound to pRb (Figure 2). Consequently,the cell cycle is brought to a halt at the G1 phase,allowing DNA repair to take place. P21 also inhi-bits proliferating cell nuclear antigen (PCNA), anauxiliary factor for DNA polymerases and ",facilitating loading of the polymerases onto DNAtemplates and increasing their activity in bothDNA replication and repair. Inhibition of PCNAby p21 is probably due to its ability to mask ele-ments on PCNA required for the binding of com-ponents of the polymerase assembly [42].Interestingly, PCNA-dependent DNA replicationis inhibited [43,44], whereas PCNA-dependentnucleotide excision-repair is not affected [4547].

Once the G1/S checkpoint is passed, replicationoccurs normally, and p53 acts as a second regula-tor to avoid re-replication [48]. Failure to exert theS phase control leads to genetic instability [48].

Role of p53 in cell cycle arrest at theG2/M checkpointP53 is also involved in cell cycle regulation at theG2/M restriction point [49] by a mechanism invol-ving mainly the inhibition of CDK1 (also calledcdc2), a cyclin-dependent kinase required to enterthe mitosis phase [50]. An intact p53 C-terminaldomain is necessary to induce G2 arrest [51].

In normal cells, the complex CDK1/cyclin B [52]determines G2/M progression [53]. At the end ofthe G2 phase, CDK1/cyclin B complex is phos-phorylated by CAK (CDK-activating kinase),becomes active and allows the cell to progressinto its mitotic phase.

In damaged cells, p53 inhibits CDK1 through atleast three mechanisms: (1) p53 transcriptionallyactivates three inhibitors of CDK1: GADD45,p21Cip1/Waf1 and 14.3.3 [54] (Figure 3). GADD45was found to directly inhibit the activity of theCDK1/cyclin B complex by physical interactionwith CDK1 but not with cyclin B [55]. P21 is a uni-versal CDK/cyclin inhibitor essential for control-ling the G2 checkpoint [56]. 14.3.3 is needed toprevent mitotic catastrophe after DNA damage.It anchors CDK1 in the cytoplasm where it isunable to phosphorylate substrates required forentry into mitosis [57]. (2) P53 can regulate thecell cycle checkpoint, not only via transcription,but also by interaction with CDK1, binding to thecatalytic sub-unit of CDK1/cyclin B kinase [58]. (3)P53 also negatively regulates CDK1 transcriptionvia the NF-Y transcription factor [59].

However, p53 is also implicated in the G2/Mcheckpoint by its ability to inhibit DNA topoi-somerase II alpha gene expression [60]. Topoi-somerase II allows the higher order compactionof chromatin, required to form a mitotic chromo-some [61]. Inhibition of topoisomerase II blockscells in G2 because the chromatin is not condensedand decatenated [62].

Figure 2. Role of p53 in the G1/S cell cycle checkpoint. CdK, cyclin-dependent kinase; E2F, E2 promoter binding factor; DP, dimerisation

partner of E2F; GADD45, growth arrest and DNA damage inducible gene; pRb, retinoblastoma protein; PCNA, proliferating cell nuclear

antigen



A study carried out by Minemoto et al. [63] alsosuggests a relationship between p53 and ubiquitin-mediated degradation of mitotic cyclins and possi-ble interactions between the G2-DNA damagecheckpoint and the mitotic checkpoint.

Role of p53 in DNA repairP53 activates genes like GADD45 implicated inDNA repair by stimulating base excision repairvia PCNA [64,65]. GADD45 stimulates DNA exci-sion in vitro and inhibits entry of cells into S phase[66]. GADD45 also recognises an altered chroma-tin state and modulates accessibility to cellularproteins [67] (Figure 2).

But the exact mechanism of p53 in DNA repairis still unclear. P53 could induce the recruitment ofnucleotide excision repair factors XPC (xerodermapigmentosum group C) and TFIIH to damagedDNA [68]. Therefore, p53 seems to play a signifi-cant role in damage recognition and subsequentassembly of repair machinery during global geno-mic repair, with an in vivo requirement for p53 inregulating the base excision repair response [69].Moreover, evidence was also found of physicaland functional interactions between p53 andDNA polymerase alpha-primase, an enzymeinvolved in DNA repair after damage provokedby stress [70]. P53 could also act as a chromatin

accessibility factor, mediating UV-induced globalchromatin relaxation prior to global nucleotideexcision repair; a fundamental component ofDNA repair, which plays a direct role in rectifyingDNA damage [71].

P53 also exhibits 30-to-50 exonuclease activityand it has been suggested that p53 might act asan external proofreader [72]. This activity seemsto be intrinsic to wild-type p53, being associatedwith the central core DNA-binding domain ofthe p53 protein structure [73]. P53, through itsexonuclease activity, could be actively involvedin detecting mismatched nucleotide pairs andin effecting nucleotide excision repair and recom-bination. Further evidence was added in amore recent study demonstrating that the exonu-clease role of p53 primarily consists of excis-ing mismatched nucleotides from DNA, soenhancing the DNA replication fidelity of poly-merase [74].

This newly discovered DNA repair role of p53,significantly extends the importance of p53 inmaintaining genomic integrity as the guardian ofthe genome. Some authors, however, have recentlyquestioned these conclusions, since p53-associated30-50 exonuclease activity has been detected innuclear and cytoplasmic compartments of cellsand also because wild-type p53 in its non-induced

Figure 3. Regulation of the G2/M checkpoint by p53. CDK1, cyclin-dependent kinase 1; P, phosphorylation; GADD45, growth arrest and

DNA damage inducible gene; CAK, CDK-activating kinase



state in the cytoplasm appears to exert 30-50

exonuclease activity [75]. Therefore the preciserole of p53 in DNA repair still remains unclear.

Role of p53 in apoptosisApoptosis is a genetically programmed process,which controls many biological events, includingcell homeostasis, tissue morphogenesis, develop-ment of the immune system and aging. It is alsoan important cellular defence against cancer andviral infection. P53 plays a critical role in apoptosis.The mechanisms by which p53 modulates activa-tion of apoptosis remain imperfectly understoodbut seem to be localised in the C-terminal basic reg-ulation domain between residues 364393 and theproline-rich domain between 6491 in the p53 pro-tein [76]. P53 is able to transactivate the transcrip-tion of range of proapoptotic genes including forexample, Bax [77] and CD95/FAS [78] (Table 2).

P53 appears to activate the apoptotic machineryby triggering translocation of Bax to mitochondria,with a concomitant release of cytochrome c frommitochondria into the cytosol. The cytochrome crelease mechanism induced by p53 was shown todepend on the presence of cytosolic Bax [79]. Inaddition p53 triggered apoptosis in oncogene-expressing fibroblasts seems to be inducedthrough the induction of Noxa and by mitochon-drial Bax translocation. At a cellular level,enforced expression of p53 is sufficient to induceBax translocation and cytochrome c release [80].This then causes activation of several caspasesincluding caspase 8, which might amplify theapoptotic signal by activating caspase 9 and itsdownstream caspases [81]. Cytochrome c bindsto APAF-1 (apoptotic protease activating factor 1)protein and participates in activation of the cas-pase cascade leading to DNA cleavage [82,83]and consequently to cell death. Nakano andcolleagues suggested that PUMA plays a role inmediating p53-induced cell death through thecytochrome c/APAF-1-dependent pathway [84].

P53 can also directly transactivate the transcrip-tion of caspase-1 [85,86] and caspase-6 [87]. Noneof the members of the caspase family had beenidentified as a direct transcriptional target of p53before caspase-1. The executioner caspase 6 isdirectly regulated by p53, provoking a loweringof the cell death threshold in response to apoptoticsignals. MacLachlan et al. [87] therefore suggestedsuch a model might provide a potential mechan-

ism for lowering cell-death thresholds and be ofpractical use in chemosensitisation.

A fraction of p53 seems to translocate directly tothe mitochondria of apoptosing tumor cells. Tar-geting p53 to mitochondria is sufficient to launchapoptosis. P53 translocation to mitochondria couldoccur in vivo in irradiated thymocytes. Further, p53could directly induce permeabilisation of the outermitochondrial membrane by forming complexeswith protective BclXL and Bcl-2 proteins, resultingin cytochrome c release. Tumor-derived transacti-vation-deficient mutants of p53 concomitantly losethe ability to interact with BclXL and to promotecytochrome c release. This implies that mutationsmight represent double-hits by abrogating thetranscriptional and mitochondrial apoptotic activ-ity of p53 [88].

During apoptosis, p53 can also inhibit the tran-scription of anti-apoptotic genes such as Bcl-2,which protect cells against programmed death[89]. Bcl-2 and Bax, which have opposite effects,belong to the same family. Bcl-2 associates withBax, and it is hypothesised that the response of acell to a death signal is determined by the ratioof Bcl-2 to Bax [90].

The difference between apoptosis and growtharrest mediated by p53 could result in modulationof the cellular response to p53 by proliferative sig-nals. Studies of p53 signaling upon -irradiation ofprimary mouse lymphocytes demonstrated thatG(0) lymphocytes rapidly went into p53-depen-dent apoptosis whereas stimulated lymphocyteswent into a p53-dependent, p21-mediated growtharrest. The switch in p53 response upon stimula-tion was not the result of a switch in transcrip-tional activation of major p53 target genes butwas mediated by proliferative signals from thecells [91].

Activation of p53The post-translational modifications seen aboveonly represent a partial picture of p53 functionalregulation. Final p53 activation is a complex me-chanism also affected by a range of proteins thateither enhance or suppress p53 activation throughdifferent pathways in response to cell stress.

Many cellular proteins are able to augment p53activity under stress conditions (Figure 4). Theprincipal p53 activation pathway appears to bevia HDM2 (main p53 inhibitor) inhibition leadingto an inhibition of p53 degradation. For example,



activation of p53 under cell stress conditions dep-ends to a great extent on the presence of c-Abl(Abelson murine leukemia cellular oncogene)which protects p53 from the inhibitory effect ofHDM2 [9295]. P14ARF/p19ARF (also called ARFfor ADP-ribosylation factor 1), with its collaboratorCARF [96] are also strong activators of p53. ARFbinds to HDM2 and promotes its rapid degrada-tion [97]. Other cellular proteins share similarp53 activating mechanisms such as HIF-1 (hypox-ia-inducible factor 1 alpha) [98], ING (inhibitor ofgrowth family) family gene, p47ING3 [99,100].

There also exist a number of other p53 activationpathways unrelated to HDM2 such as BRCA-1(breast cancer 1 gene) [101104] and PARP-1(poly(ADP-ribose) polymerase-1) [105,106].

Inhibition of p53HDM2 (often called MDM2: murine double min-ute) probably constitutes the major inhibitor ofp53 (Figure 4). It binds to the transactivationdomain of p53 whilst the protein is localised inthe nucleus (Figure 1). Although the N-terminaldomain of p53 contains the primary docking siteof HDM2, a second HDM2 binding interface with-in the linker region of the core domain of p53 hasbeen described [107].

HDM2 inhibits the ability of p53 to activate tran-scription of its downstream target genes by two

known mechanisms: (1) binding directly to thetransactivation domain of p53 and blocking itsinteraction with TBP-associated factors TAFII70and TAFII31 (see details above) [23,24,108]; (2) tar-geting p53 for degradation via the ubiquitin-26Sproteasome pathway [109]. HDM2 functions as aubiquitin ligase for p53 [110] mediating ubiquiti-nation of p53 and allowing it to be recognisedand degraded by the proteasome [111]. HDM2-mediated p53 proteasomal degradation seems tobe regulated by NAD(P)H quinone oxidoreductase1 (NQO1) [112,113] and the HDM2/p53 complex isexported from the nucleus using HDM2 nuclearexport signal (NES). Nuclear export of p53 seemsto be necessary before the protein can be degraded[114]. Regulatory mechanisms exist to controlexport of p53 into the cytoplasm, such as the exis-tence of two NES on the p53 protein itself [115].But in fact, nuclear degradation of p53 could occurduring down-regulation of the p53 response afterDNA damage. And local p53 degradation withinthe nucleus could provide a tighter and faster con-trol route during the down-regulatory phase [116].

HDM2 also inhibits p300-mediated p53 acetyla-tion by forming a ternary complex with p53 andp300 [117], significantly repressing activation ofp53 transcriptional activity without apparentlyaffecting the level of p53 [118]. Acetylation ofp53 inhibits its ubiquitination by HDM2 [119].

Figure 4. P53 protein can be positively and negatively regulated by a large number of cellular proteins



P53 transactivates HDM2 transcription afterbeing activated, by recruiting a TRRAP/acetyl-transferase complex to the HDM2 gene to activateits transcription [120] (Figure 1). Interestingly,HDM2 seems to be required for rapid turnoverof p53 [121]. Because HDM2 is transcriptionallyactivated by p53, this degradative pathway maycontribute to the maintenance of low p53 concen-trations in normal cells [111], thus providing anauto-regulatory feedback loop [122].

Inhibiting the p53-HDM2 interaction withsynthetic molecules is a promising approach foractivating p53 and could lead to p53-mediatedcell-cycle arrest or apoptosis (for review see, [123]).

Other cellular proteins are also able to inhibitp53 activity such as HDMX [124,125], phosphati-dylinositol 3-OH-kinase (Akt) [126].

HUMAN TUMOR SUPPRESSOR P53AND DNA VIRUSESSome viruses are associated with human cancers(Table 1), but their real roles in the developmentof cell transformation are still unclear. Forinstance, studies of many viruses revealed theirability to nonspecifically induce cytogeneticdamage to their host cell chromosomes. For exam-ple, the oncogenic adenoviruses, HSV and HCMVhave been found to cause non-random, site-speci-fic chromosomal damage (for review, see [127]). Inthese different cases, if following a break in thechromosome, there is some DNA repair, then thecell may survive, but may harbor specific muta-tions, which could favor cancer development,aggravated by damaged tumor suppressors.

DNA tumor viruses typically infect resting cellsand induce transition from G0 or G1 into the S-phase of the cell cycle. This phase is permissivefor virus replication, presumably because hostnucleotide pools and proteins involved in DNAsynthesis are necessary for viral replication. Toenter S-phase, cells must traverse the p53-pRbcheckpoint, and induce E2F activity. However,forced entry into S-phase induced by E2F activityprovokes apoptosis, and so these viruses also needto suppress apoptosis to replicate efficiently. AllDNA viruses implicated so far in cell transforma-tion have been shown to encode viral proteins cap-able of inducing inactivation of tumor suppressorprotein p53 functions (Table 1). A similar viralmechanism, based on proteinprotein interaction,is also known to block the functions of pRb, allow-

ing simultaneously the activation of E2F activityand inhibition of apoptosis.

Inactivation of p53 is critical for the replicationof DNA tumor viruses and the main mechanismsemployed to achieve this are largely based on thebinding of viral proteins to p53. Binding of viralprotein in most cases reduces the transcriptionalactivation function of p53. In the following para-graphs, the different strategies used by DNAviruses to abrogate p53 functions are summarised.

Small DNA virusesAlthough adenoviruses are not known to be asso-ciated with tumorigenesis in humans, somehuman adenovirus serotypes can directly inducetumours in rats or hamsters, and all serotypestested can transform cultured rodent cells [128].Two viral genes E1A and E1B were shown to beboth necessary and sufficient for transformation[129]. Transformation of primary rodent cells byadenovirus E1A and E1B oncogenes is a two-stepprocess, where E1A-dependent induction of proli-feration is coupled to E1B-dependent suppressionof apoptosis.

The adenovirus E1B gene encodes two proteins,the E1B55K protein and the E1B19K. E1B55K pro-tein forms a stable complex with p53 in vitro andin vivo [130,131] and inhibits p53-mediated tran-scription activation [132] by binding to the ami-no-terminal transactivation domain of p53 [131].E1B19K protein does not inhibit p53-mediatedtransactivation but alleviates p53-mediated tran-scriptional repression. P53 may induce apoptosisnot through the activation of transcription butrather through transcriptional repression and theE1B19K protein may overcome p53-mediatedapoptosis by alleviating p53-mediated transcrip-tional repression [133].

Inhibition of p53 can also depend upon indirectand sometimes more subtle interactions. E1B55Kprotein physically interacts with the P/CAF-p53complex suggesting that the E1B55K protein inhi-bits P/CAF acetylase function on p53 by prevent-ing enzymesubstrate interaction [134]. Withoutacetylation, p53 remains in an inactivated stateand fails to function.

Adenovirus E4orf6 is also able to interact withp53 within amino acids 318360 of the carboxy-terminal domain of p53 but, unlike E1B55K,E4orf6 blocks p53-stimulated transcription byinterfering with the ability of the amino-terminal



domain of p53 to contact TAFII31. E4orf6 proteininteracts at a site on p53 distinct from the domainthat binds to TAFII31 but nevertheless inhibitsp53TAFII31 interaction [135].

Inhibition of the transcriptional activity of p53 isnot the only consequence of viral proteins bindingto p53; such interaction can also provoke thedegradation of p53. E1B55K and E4orf6 proteinsboth bind independently to p53 and together pro-mote its rapid degradation [136] via a novelmechanism involving a cullin-containing E3 ubi-quitin ligase [137].

Even though E1A is considered responsible forp53 protein stabilisation [138], E1A protein couldalso repress p53-mediated transactivation, withoutprovoking a significant effect on the DNA-bindingcapacity of p53. Apparently, E1A causes increasedhomo- or hetero-oligomerisation of p53, whichmight result in inactivation of the transcriptionactivation domain of p53, blocking its tetramerisa-tion. Additionally, transfectant cells stably expres-sing E1A lose their ability to arrest the cell cycle atthe G1 stage following DNA damage, suggestingthat E1A can abolish the normal biological functionof p53 [139]. Moreover, p300/CBP and TBP,required for p53 activity, have been identified asfunctional targets of E1A in transcriptional regula-tion, and may therefore provide a pathway bywhich E1A can exert its transforming activity [140].

The SV40 is a macaque polyomavirus. It has notbeen associated with tumors in its natural host butit is known to induce tumors in new-born rodents.It was iatrogenically introduced into millions ofpeople in North America and Europe between1955 and 1962 through SV40-contaminated poliovaccines [141]. SV40 has also been associatedwith a number of human cancers and there is con-troversy as to whether the virus contributes totumorigenesis. The viral sequence encoding thelarge T antigen (Tag) is always expressed in trans-formed cells, and Tag is sufficient to transformrodent stem cells. Tag is a multifunctional DNA-binding protein that initiates viral DNA replica-tion. Tag is both a transcriptional repressor andactivator, composed of three independent domainsthat contribute to transformation. The first trans-forming region is a J domain (present in membersof DNA J-family of chaperones). The second onegoverns interaction with the pRb and relatives.The third transforming region allows interactionwith p53. All three regions are required for trans-

formation in most cell types [142144]. SV40 Tagbinds to p53 and inhibits its capacity to activatetranscription. The viral protein could prevent p53from binding to the ribosomal gene cluster (RGC)site [145] on cellular DNA, which contains p53recognition sequences including p53-binding frag-ments, and furthermore wild-type p53 could sti-mulate transcription of reporter genes with RGCin their 50 region [146]. Tag, under certain condi-tions, may also repress p53-dependent transcrip-tion by a mechanism in which the transactivationdomain of p53 is abrogated while DNA binding isunaffected [147]. Simultaneously, p53 has beenshown to stabilise Tag, thus augmenting its onco-genic potential [148]. Tag can also repress p53-mediated transcription by blocking the interactionof p53 with transcription coactivators such asp300. CBP and p300 have been shown to interactwith SV40 large T antigen inducing an alterationof the phosphorylation states of both proteinsand consequently inhibiting their transcriptionalactivities [149]. P300 binds to a carboxyl-terminalfragment of large T antigen which contains itsp53 binding domain, but the implications for p53functions are not yet established [150]. SV40 largeT antigen also represses p53-mediating transcrip-tion by deleteriously affecting the assembly or con-formation of the basal transcription machineryrequired for p53 functions. Large T antigen couldalso interact with both TBP and several TAFs [151].

Some studies have reported the presence of Tag,and of Tag/p53 complexes, in SV40-associatedhuman tumors [152,153]. More work needs to bedone to accept the hypothesis that SV40 causescancers in humans (for review, see [154]).

HPVs are small DNA viruses clearly associatedwith the induction of cancer. Clinical and epide-miological studies have implicated HPV infectionin the development of cervical carcinomas. Thehigh-risk HPVs (most notably HPV16 and 18) areassociated with high-grade squamous intraepithe-lial lesions and invasive cervical carcinomas,whereas the low-risk types are found more oftenin low-grade lesions [155]. In the majority of pri-mary cervical carcinoma and cervical tumor celllines studied, the viral genomes of high-risk HPV(16 and 18) types are integrated into the host-genome, allowing active expression of E6 and E7genes. The transforming activities of E6 werefirst revealed when it was shown that efficientimmortalisation of primary human fibroblasts or



keratinocytes requires the combination of E6 andE7 [156]. E6 protein binds to p53 and inhibits tran-scriptional activation, inducing changes within thep53 protein which result both in inhibition of DNAbinding and in dissociation of p53 protein pre-viously bound to the DNA [157].

It has become increasingly clear that many smallDNA tumor viruses have evolved complexmechanisms to functionally inactivate the p53.But the mechanism employed by HPV E6 proteinsto inactivate p53 functions is different; eventhough Tag, E1B55K and E6 proteins can eachform complexes with p53 that lead to functionalinactivation, these interactions have differentimpacts on the stability of the p53 [158]. SV40Tag and E1B55K are able to provoke an extendedmetabolic half-life of p53 [159,160], caused bysequestration of p53 into stable but nonfunctionalcomplexes. The high-risk HPVs have developed aunique strategy to inactivate the p53 by accelerat-ing its proteolytic degradation. MechanisticallyHPV-16 E6 accelerates p53 degradation by forminga complex with, and reprogramming, the cellularubiquitin-protein ligase E6AP [161,162]. TheE6/E6AP complex acts as a p53-specific ubiqui-tin-protein ligase [163] (for review, see [164]).

The ability of E6 to induce p53 degradation viaubiquitination would be independent of the sixterminal lysine residues in p53 which had pre-viously been identified as playing an importantrole in effective ubiquitination and degradationof p53 mediated by HDM2. E6 could induce ubi-quitination of p53 using an alternative mechanismto that employed by HDM2 in promoting p53degradation [165]. However, an earlier studyshowed that HPV18 E6 mediated-inhibition oftranscriptional activity of p53 could be indepen-dent of E6 induced degradation [157].

E6 protein of HPV16 also binds to three regionsof both CBP and p300, resulting in abrogation ofCBP/p300 transcriptional co-activator functions.E6 may also suppress p53 activation via theCBP/p300 complex quite independently from itsknown ability of binding to p53 and removing cel-lular p53 via the proteasomal degradation path-way [166,167].

HBV produces chronic infections of the liverleading to cirrhosis and hepatocellular carcinoma.The X protein (HBx) of HBV, which is able totransactivate viral and cellular genes by interact-ing directly with cellular transcription factors,

might be involved in the development of the hepa-tocarcinogenesis. It is capable of neoplasticallytransforming rodent cells [168], and causing HCCin transgenic mice [169]. HBx is a multifunctionalprotein that can interact with p53. However, theexact molecular events involved in liver carcino-genesis remain to be elucidated. HBx binds tothe C-terminus of p53 and partially sequesters itin the cytoplasm, preventing interaction betweenp53 and cellular transcription factors, thereby inhi-biting p53 action on transcriptional machinery[170,171]. A wide range of p53 functions may con-sequently be affected including the down-regula-tion of p53-induced DNA repair mechanismswhich would lead to accumulation of geneticdefects and increasing carcinogenesis [172].

Moreover HBV core protein (HBc) functions as arepressor of human p53 gene expression by inhi-biting the promoter activity in the human livercell line HepG2, and also acting synergisticallywith HBx protein in this repressive effect [173].

Recently, transgenic mouse models of HBV-associated hepatocellular carcinoma were used inorder to understand better the role of HBV in liverdisease and to highlight the importance of viralgene products in the cell cycle control checkpointderegulations in the hepatocytes [174].

Large DNA virusesHerpesviruses are widespread within the world-wide population. Some of them are involved incell transformation in vitro and could also be impli-cated in human cancer development. They seem todevelop similar strategy consisting in p53 functioninactivation but some of them are also capable ofdeveloping many other mechanisms.

HCMV is an important human pathogen thatcauses congenital birth defects and severe oppor-tunistic diseases. It has also been suspected tohave oncogenic properties. Several regions ofHCMV genome have transforming ability, includ-ing some of the IE genes. The etiological associa-tion between HCMV and human cancer remainsan enigma. The ubiquitous nature of HCMV with-in the population would lead one to expect muchhigher incident rates for each of the malignanciessupposedly linked to the virus. In light of this, ithas been suggested that HCMV may act as a co-etiologic agent in the development of tumorsunder conditions not well understood at this point.HCMV may indirectly contribute to oncogenesis



through a hit-and-run mechanism [175]. Essen-tially it would act as a tumor promoter, perhapsby causing genetic instability or by preventingcells from undergoing apoptosis (for review, see[176]). HCMV IE-86 protein can reduce p53 tran-scriptional capacity by direct interaction [177],though without affecting G1 checkpoint function[178]. The oncogenic potential of HCMV was ori-ginally established on the basis of in vitro studieson hamster fibroblasts [179]; recently, HCMVnucleic acids and proteins were found specificallylocalised to neoplastic cells in human colorectalpolyps and adenocarcinomas. Virus infectioncould induce important oncogenic pathways incolon-cancer cells [180].

HHV-6, a T-lymphotropic virus highly endemicin humans, is the etiologic agent of the commonfebrile childhood exanthem subitum. It has alsobeen suggested to play a role in the developmentof lymphoproliferative disorders and especially inHodgkins disease [181]. The DR7 viral gene is ableto transform NIH3T3 cells and is responsible forthe development of tumours in nude mice. Theprotein DR7 encoded by this gene is also able tobind to p53 and to inhibit the p53-mediated tran-scriptional activation [182].

EBV is a -herpesvirus, which is the causativeagent of infectious mononucleosis, and whichestablishes a life-long persistent infection in over95% of the human adult population world-wide.It seems to be the most highly transforming mem-ber of the herpesvirus family, efficiently immorta-lising human B-lymphocytes in vitro. It isassociated with the development of several malig-nancies, including Burkitts lymphoma, nasophar-yngeal carcinoma, Hodgkins disease and T-celllymphomas. EBV BZLF1 protein, which initiatesthe switch of latent to lytic EBV infection, interactsdirectly in vitro and in vivo with the carboxy-term-inal portion of p53, and it is possible this may bepart of the latent to lytic viral infection pathway[183]. BZLF1 affects p53 post-translational modifi-cation and is known to inhibit p53 transcriptionalfunction leading some researchers to propose theexistence of an indirect mechanism involving sup-pression of TBP expression [184]. But this virus iscapable of developing a wide range of differentmechanisms in addition to p53 inactivation toinduce cell transformation. For example, the latentmembrane protein 1 (LMP1) plays a central part inthe transforming process by mimicking members

of the family of tumor necrosis factor (TNF) recep-tors, transmitting growth signals [185].

HHV-8 is also a human -herpesvirus that playsa role in the development of Kaposis sarcomalesions, primary effusion lymphoma and multi-centric Castlemans disease, which is an atypicallymphoproliferative disorder [186]. Several HHV-8 proteins are known to be oncogenic such asLANA and v-IRF-1.

V-IRF-1 protein from HHV-8, which acts as aregulator for the expression of several HHV-8genes, interacts and inhibits the transcriptionalco-activator p300 [187], thus provoking indirectinhibition of p53 functions.

Moreover, K8, an early protein encoded by HHV8also binds to p53 and functions in the recruitment ofp53 to the promyelocytic leukemia protein (PML)bodies which are nuclear sites for both input viralgenome deposition and immediate-early (IE) genetranscription during infection [188].

Finally, ORF50 protein which is a viral transcrip-tional activator of several early and late genes ofHHV8, also represses the transcriptional activityof p53 and the p53-induced apoptosis [189].

CONCLUSIONAs seen in this review, the p53 tumor suppressororchestrates a number of important genesinvolved in cell-cycle control and apoptosis. Itseems to be one of the most important moleculesin regulating cell growth and in protecting cellsfrom malignant transformation. When p53 func-tion is impaired, its ability to preserve genomicintegrity is compromised and this may result inan increase in mutations on the p53 gene and con-tribute to progression towards a malignant pheno-type. However, the p53 signaling pathway alsoconnects with tumor suppressors and viral onco-genes known to influence cell cycle machinery.Alterations in components either upstream ordownstream of p53 may therefore be analogousto inactivation of p53 itself, leading to deregulationof cell cycle control, genomic instability and devel-opment of cancer.

The DNA viruses implicated in the develop-ment of human cancers exhibit a similar mechan-ism aimed at inhibiting both p53 and pRb. The factthat transforming viral proteins form complexeswith two major tumor suppressor proteins sug-gests a remarkable case of convergent evolution.Induction of apoptosis by p53 in response to viral



infection may explain why DNA tumor viruseshave evolved such similar approaches to disablep53 functions [190].

Researches conducted on interactions betweenp53 and viral oncoproteins provided significantinformation on human cancer development. Thepursuit of the studies on the viral oncogenesshould allow the discovery of new factors impli-cated in the control of the cell cycle, and couldbe helpful in the design of new anti-tumoral drugs.For example, Cassetti et al. have developed Vene-zuelan equine encephalitis (VEE) virus repliconparticle (VRP). The fusion proteins were mutatedat four amino acid positions to inactivate theironcogenic potential, in order to stimulate theimmune system against E6 and E7 [191].

The question of why viruses need to inactivatep53 has not been formally resolved, although sup-pression of G1/S checkpoint and apoptosis appearlikely to be critical. Small DNA viruses depend onhost cell machinery to replicate their own DNAand, clearly, a protein that blocks S-phase and cap-able of inducing apoptosis could inhibit virusreplication.

Nevertheless, even if p53 is mutated in morethan 50% of primary human tumors and inacti-vated by interaction with viral oncogenic proteinsor other molecules, this alone is insufficient forthe development of cancer, despite being of greatimportance in the multistep carcinogenic process.Where necessary, other members of the p53 family,p63 and p73, can provide a second control pathwayfor the protection and repair of stressed cells.

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