a-comparative-approach-to-characterize-landscape-host-pathogen

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Institut Pasteur 

We have characterized Xenopus ISWI, a catalytic subunit of a family of chromatin-remodeling complexes. We show that ISWI is expressed constitutively during development but poorly expressed in adult tissues except oocytes which contain a large store of maternal protein. We further analyzed its localization both in vivo and in vitro in Xenopus cell cycle extracts and identified that ISWI binds to chromatin at the G1-S period. However, its association to chromatin does not require ongoing DNA replication. Immunodepletion of ISWI has no effect on either sperm chromatin decondensation or the kinetics and efficiency of DNA replication. Nucleosome assembly also occurs in ISWI-depleted extracts, but nucleosome spacing is disturbed. From these results, we conclude that ISWI is not necessary for sperm chromatin decondensation and the accelerated rates of DNA replication that characterize early development.

Expression of ISWI and its binding to chromatin during the cell cycle and early development.

Nous avons caract&eacute;ris&eacute; Xenopus ISWI, une sous-unit&eacute; catalytique d&#39;une famille de complexes de remodelage de la chromatine. Nous montrons que ISWI est exprim&eacute;e constitutivement au cours du d&eacute;veloppement mais mal exprim&eacute;e dans les tissus adultes sauf les ovocytes qui contiennent un grand magasin de prot&eacute;ine maternelle. Plus loin, nous avons analys&eacute; sa localisation in vivo et in vitro dans des extraits de cycle cellulaire Xenopus et identifi&eacute;s que ISWI se lie &agrave; la chromatine &agrave; la p&eacute;riode G1-S. Cependant, son association &agrave; la chromatine n&#39;exige pas la r&eacute;plication de l&#39;ADN en cours. Protocole de ISWI n&#39;a aucun effet sur soit d&eacute;condensation de la chromatine de sperme ou la cin&eacute;tique et l&#39;efficacit&eacute; de la r&eacute;plication de l&#39;ADN. Assembl&eacute;e nucl&eacute;osome se produit &eacute;galement dans les extraits ISWI appauvri, mais espacement nucl&eacute;osome est perturb&eacute;. D&#39;apr&egrave;s ces r&eacute;sultats, nous concluons que ISWI n&#39;est pas n&eacute;cessaire pour la d&eacute;condensation de la chromatine de sperme ainsi que les taux acc&eacute;l&eacute;r&eacute;s de la r&eacute;plication de l&#39;ADN qui caract&eacute;risent le d&eacute;but du d&eacute;veloppement.

Expression de ISWI et sa liaison &agrave; la chromatine au cours du cycle cellulaire et du d&eacute;veloppement.

Noi abbiamo caratterizzato Xenopus ISWI, una subunit&agrave; catalitica di una famiglia di complessi di rimodellamento della cromatina. Mostriamo ISWI &egrave; espresso costitutivamente durante lo sviluppo, ma scarsamente espressi in tessuti adulti tranne gli ovociti che contengono un grande negozio di proteina materna. Abbiamo ulteriormente analizzata la localizzazione sia in vivo che in vitro in Xenopus ciclo cellulare estratti e identificato ISWI si lega alla cromatina presso il periodo G1-S. Tuttavia, l&#39;associazione di cromatina non richiede la replicazione del DNA in corso. Immunodepletion di ISWI non ha alcun effetto su decondensazione della cromatina degli spermatozoi o la cinetica e l&#39;efficienza di replicazione del DNA. Assemblaggio del nucleosoma si verifica anche in estratti ISWI-esaurito, ma la spaziatura nucleosoma &egrave; disturbato. Da questi risultati, concludiamo che ISWI non &egrave; necessaria per la decondensazione della cromatina degli spermatozoi e le tariffe accelerate della replicazione del DNA che caratterizzano lo sviluppo precoce.

Espressione di ISWI e relativa associazione a cromatina durante il ciclo cellulare e lo sviluppo precoce.

Cervical carcinomas are most frequently associated with human papillomaviruses (HPV), whose E6 and E7 oncogenes products induce cellular immortalization. The papillomavirus E2 protein is a transcription factor, which represses the expression of the viral oncogenes, and activates viral DNA replication during the vegetative viral cycle. This protein is specifically inactivated in HPV18-associated carcinoma cells, suggesting that E2 functions prevent carcinogenic progression. Indeed, ectopic expression of E2 in cervical carcinoma cells strongly inhibits cell proliferation. Here we show that above a threshold level of expression, the E2 protein induces apoptosis, independently of other viral functions. The amino-terminal domain is responsible for this apoptotic activity, but surprisingly with no involvement of its transcriptional functions. The death pathway triggered by E2 relies on activation of the initiator caspase 8, specific of the extrinsic pathway of apoptosis. E2 itself is cleaved by caspases during cell death, providing an example of an apoptotic inducer that is itself a target for caspase processing. The autonomous proapoptotic activity of HPV18 E2 described here may counteract the proliferative functions of viral oncogenes, and renders the inactivation of E2 crucial for carcinogenic progression.

Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein.

Carcinomes du col ut&eacute;rin sont plus souvent associ&eacute;s aux virus du papillome humain (VPH), dont les produits oncog&egrave;nes E6 et E7 induisent immortalisation cellulaire. Le virus du papillome prot&eacute;ine E2 est un facteur de transcription, qui r&eacute;prime l&#39;expression des oncog&egrave;nes virus et active la r&eacute;plication de l&#39;ADN virale durant le cycle v&eacute;g&eacute;tatif virale. Cette prot&eacute;ine est sp&eacute;cifiquement inactiv&eacute;e dans les cellules de carcinome associ&eacute;e &agrave; HPV18, sugg&eacute;rant que E2 fonctions pr&eacute;venir canc&eacute;rog&egrave;ne progression. En effet, l&#39;expression ectopique de E2 dans les cellules de carcinome cervical inhibe fortement la prolif&eacute;ration cellulaire. Nous d&eacute;montrons que dessus d&#39;un niveau d&#39;expression, la prot&eacute;ine E2 induit l&#39;apoptose, ind&eacute;pendamment des autres fonctions virales. Le domaine amino-terminale est responsable de cette activit&eacute; apoptotique, mais &eacute;tonnamment ne comportant aucune participation de ses fonctions de transcriptional. La voie de la mort provoqu&eacute;e par l&#39;E2 s&#39;appuie sur l&#39;activation de la caspase initiatrice 8, sp&eacute;cifique de la voie extrins&egrave;que de l&#39;apoptose. E2 elle-m&ecirc;me est cliv&eacute; par les caspases durant la mort cellulaire, fournissant un exemple d&#39;un inducteur de l&#39;apoptose qui est elle-m&ecirc;me une cible pour le traitement des caspases. L&#39;activit&eacute; proapoptotique autonome de E2 HPV18 d&eacute;crite ici peut contrecarrer les fonctions prolif&eacute;ratives des oncog&egrave;nes virus et restitue l&#39;inactivation de E2 crucial pour progression canc&eacute;rig&egrave;ne.

Transcription ind&eacute;pendant d&eacute;clenchement de la voie extrins&egrave;que de l&#39;apoptose par la prot&eacute;ine E2 de virus du papillome humain 18.

Carcinomi cervicali sono pi&ugrave; frequentemente associati con papillomavirus umano (HPV), cui prodotti di oncogeni E6 ed E7 inducono immortalization cellulare. Il papillomavirus proteine E2 &egrave; un fattore di trascrizione, che reprime l&#39;espressione degli oncogeni virali e attiva replicazione del DNA virale durante il ciclo vegetativo virale. Questa proteina &egrave; specificamente inattivata in cellule di carcinoma associato HPV18, suggerendo che funzioni E2 prevenire la progressione cancerogena. In effetti, espressione ectopica di E2 in cellule di carcinoma cervicale fortemente inibisce la proliferazione cellulare. Qui vi mostriamo che di sopra di un livello di soglia dell&#39;espressione, la proteina E2 induce l&#39;apoptosi, indipendentemente da altre funzioni virali. Il dominio amino-terminale &egrave; responsabile di questa attivit&agrave; apoptotic, ma sorprendentemente con nessun coinvolgimento delle sue funzioni di transcriptional. Il pathway di morte innescato da E2 si basa sull&#39;attivazione dell&#39;iniziatore caspase 8, specifico del percorso estrinseco dell&#39;apoptosi. E2 si &egrave; fenduta dai caspases durante la morte cellulare, fornendo un esempio di un induttore apoptotic che &egrave; di per s&eacute; una destinazione per l&#39;elaborazione di caspase. L&#39;attivit&agrave; autonoma Miselli HPV18 E2 qui descritto pu&ograve; contrastare le funzioni proliferative degli oncogeni virali e rende l&#39;inattivazione di E2 cruciale per progressione cancerogena.

Trascrizione-indipendente innesco del pathway estrinseco dell&#39;apoptosi da papillomavirus umano 18 proteina E2.

More than 90% of cervical carcinomas are associated with human papillomavirus (HPV) infection. The two viral oncogenes E6 and E7 play a major role in transforming the cells by disrupting p53- and pRb-dependent cell cycle checkpoints. A hallmark of HPV-associated cervical carcinoma is loss of the expression of the viral E2 protein, often by disruption of E2-encoding gene. We showed previously that reintroduction of E2 in HPV18-associated cervical carcinoma cells induces cell cycle arrest in G(1) because of the transcriptional repression of the viral oncogenes E6 and E7 and concomitant reactivation of the p53 and pRb pathways. Here we describe global gene profiling of HeLa cells expressing different HPV18 E2 mutants to study the effects of repression of the viral oncogenes. We identified 128 genes transcriptionally regulated by the viral oncogenes in cervical carcinoma. Surprisingly, E2 repressed a subset of E2F-regulated mitotic genes in an E6/E7-dependent pathway. This was corroborated by the observation that E2 delayed mitotic progression, suggesting the involvement of a mitotic pathway in HPV carcinogenesis. These mitotic genes constitute an as yet unrecognized set of genes, which were also found deregulated in other HPV-associated cervical carcinoma cell lines and therefore represent new targets for both diagnosis and therapeutic approaches in cervical cancer.

A genomic approach reveals a novel mitotic pathway in papillomavirus carcinogenesis.

Plus de 90 % des carcinomes du col ut&eacute;rin sont associ&eacute;s &agrave; l&#39;infection par le virus du papillome humain (VPH). Les deux virus oncog&egrave;nes E6 et E7 jouent un r&ocirc;le majeur dans la transformation des cellules en perturbant les points de contr&ocirc;le de cycle cellulaire d&eacute;pendant de p53 et pRb. Une caract&eacute;ristique du carcinome cervical associ&eacute;es au VPH est la perte de l&#39;expression de la prot&eacute;ine E2 virale, souvent par la rupture du g&egrave;ne codant E2. Nous avons montr&eacute; pr&eacute;c&eacute;demment que sa r&eacute;introduction de E2 dans HPV18 associ&eacute;es aux cervicales cellules carcinomateuses induit l&#39;arr&ecirc;t du cycle cellulaire en g (1) en raison de la r&eacute;pression transcriptionnelle de la r&eacute;activation virale oncog&egrave;nes E6 et E7 et concomitante des voies p53 et pRb. Nous d&eacute;crivons ici global gene profiling des cellules HeLa exprimant diff&eacute;rents mutants HPV18 E2 pour &eacute;tudier les effets de la r&eacute;pression des oncog&egrave;nes virus. Nous avons identifi&eacute; des 128 g&egrave;nes transcriptionnellement r&eacute;gul&eacute;s par les oncog&egrave;nes virus dans le carcinome du col ut&eacute;rin. &Eacute;tonnamment, E2 r&eacute;prim&eacute; un sous-ensemble de g&egrave;nes mitotiques E2F-r&eacute;gul&eacute;s dans une voie E6/E7-d&eacute;pendante. Cela a &eacute;t&eacute; corrobor&eacute; par l&#39;observation que E2 retard&eacute; la progression mitotique, sugg&eacute;rant l&#39;implication d&#39;une voie mitotique dans la carcinogen&egrave;se HPV. Ces g&egrave;nes mitotiques constituent une s&eacute;rie encore m&eacute;connue des g&egrave;nes, qui ont &eacute;galement &eacute;t&eacute; d&eacute;r&eacute;glement&eacute; dans d&#39;autres lign&eacute;es de cellules de carcinome cervical associ&eacute;es au VPH et repr&eacute;sentent donc les nouvelles cibles pour les diagnostics et les approches th&eacute;rapeutiques dans le cancer du col ut&eacute;rin.

Une approche g&eacute;nomique r&eacute;v&egrave;le une nouvelle voie mitotique dans la carcinogen&egrave;se du papillomavirus.

Pi&ugrave; del 90% dei carcinomi cervicali sono associato con l&#39;infezione da papillomavirus umano (HPV). I due oncogeni virali E6 ed E7 giocano un ruolo importante nel trasformare le cellule interrompendo i checkpoint del ciclo cellulare p53 e pRb-dipendente. Una caratteristica del carcinoma cervicale HPV-associati &egrave; la perdita dell&#39;espressione della proteina virale E2, spesso tramite rottura del gene E2-codifica. Vi abbiamo mostrato in precedenza tale reintroduzione di E2 di associato HPV18 cervicale cellule carcinoma induce arresto del ciclo cellulare in g (1) a causa della repressione trascrizionale di riattivazione virale oncogeni E6 ed E7 e concomitante delle vie p53 e pRb. Qui descriviamo global gene profiling delle cellule HeLa esprimendo diversi mutanti HPV18 E2 per studiare gli effetti della repressione degli oncogeni virali. Abbiamo identificato 128 geni transcriptionally regolamentate dagli oncogeni virali nel carcinoma cervicale. Sorprendentemente, E2 represso un sottoinsieme dei geni mitotica E2F-regolato in un percorso di E6/E7-dipendente. Ci&ograve; &egrave; stata confermata dall&#39;osservazione che E2 ritardata progressione mitotica, suggerendo il coinvolgimento di un pathway mitotica nella carcinogenesi HPV. Questi geni mitotici costituiscono un insieme non ancora riconosciuto di geni, che sono stati trovati anche liberalizzato in altre linee cellulari di carcinoma cervicale HPV-associati e rappresentano quindi nuovi bersagli per la diagnosi e approcci terapeutici in cancro cervicale.

Un approccio genomico rivela un romanzo pathway mitotica nella carcinogenesi papillomavirus.

Human Papillomavirus (HPV) E2 proteins are the major viral regulators of transcription and replication during the viral life cycle. In addition to these conserved functions, we show that E2 proteins from high risk HPV types 16 and 18, which are associated with cervical cancer, can induce apoptosis. In contrast, E2 proteins from low risk HPV types 6 and 11, which are associated with benign lesions, do not cause cell death. We show that the ability to induce apoptosis is linked to the intracellular localization of the respective E2 proteins rather than to inherent properties of the proteins. Although low risk HPV E2 proteins remain strictly nuclear, high risk HPV E2 proteins are present in both the nucleus and the cytoplasm of expressing cells due to exportin-1 receptor (CRM1)-dependent nucleo-cytoplasmic shuttling. Induction of apoptosis is caused by accumulation of E2 in the cytoplasm and involves caspase 8 activation. We speculate that disruption of the E2 gene during viral genome integration in cervical carcinoma provides a means to avoid E2-induced apoptosis and allow initiation of carcinogenesis.

Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis.

Les prot&eacute;ines humaines de Papillomavirus (HPV) E2 sont les principaux r&eacute;gulateurs virales de la transcription et la r&eacute;plication au cours du cycle de vie viral. Outre ces fonctions conserv&eacute;es, nous montrent que les prot&eacute;ines E2 de risque &eacute;lev&eacute; VPH des types 16 et 18, qui sont associ&eacute;s au cancer du col ut&eacute;rin, peuvent induire l&#39;apoptose. En revanche, prot&eacute;ines E2 de faible risque VPH des types 6 et 11 qui sont associ&eacute;es &agrave; des l&eacute;sions b&eacute;nignes, ne provoquent pas la mort des cellules. Nous montrons que la capacit&eacute; d&#39;induire l&#39;apoptose est li&eacute;e &agrave; la localisation intracellulaire des prot&eacute;ines E2 respectifs, plut&ocirc;t qu&#39;aux propri&eacute;t&eacute;s inh&eacute;rentes des prot&eacute;ines. Bien que les prot&eacute;ines de faible risque HPV E2 restent strictement nucl&eacute;aires, prot&eacute;ines de haut risque de HPV E2 sont pr&eacute;sents dans le noyau et le cytoplasme d&#39;exprimer des cellules en raison de l&#39;exportation-1 receptor (CRM1)-d&eacute;pendante la navette nucl&eacute;o-cytoplasmique. Induction de l&#39;apoptose est caus&eacute;e par l&#39;accumulation de E2 dans le cytoplasme et implique l&#39;activation de la caspase 8. Nous sp&eacute;culons que perturbation du g&egrave;ne E2 au cours de l&#39;int&eacute;gration du g&eacute;nome viral dans le carcinome du col ut&eacute;rin constitue un moyen d&#39;&eacute;viter l&#39;apoptose induite par l&#39;E2 et de permettre l&#39;initiation de la canc&eacute;rogen&egrave;se.

La navette nucl&eacute;o-cytoplasmique de prot&eacute;ines humaines de Papillomavirus E2 risque &eacute;lev&eacute; induit l&#39;apoptose.

Proteine umane di Papillomavirus (HPV) E2 sono i principali regolatori virali della trascrizione e replica durante il ciclo di vita virale. Oltre a queste funzioni conservate, ci mostra che le proteine E2 da tipi di HPV ad alto rischio 16 e 18, che sono associati con cancro cervicale, possono indurre il apoptosis. Proteine E2 da tipi di HPV a basso rischio 6 e 11, che sono associati a lesioni benigne, in contrasto, non causano la morte delle cellule. Mostriamo che la capacit&agrave; di indurre l&#39;apoptosi &egrave; legata alla localizzazione intracellulare delle rispettive proteine E2, piuttosto che alle propriet&agrave; intrinseche delle proteine. Anche se le proteine E2 di HPV a basso rischio restano rigorosamente nucleare, proteine ad alto rischio HPV E2 sono presenti in entrambi il nucleo e del citoplasma delle cellule dovuto exportin-1 receptor (CRM1) di esprimere-dipendente nucleo-citoplasmatici spola. Induzione di apoptosi &egrave; causato dall&#39;accumulo di E2 nel citoplasma e coinvolge l&#39;attivazione di caspase 8. Si ipotizzano che turbativa del gene E2 durante l&#39;integrazione del genoma virale nel carcinoma cervicale consente di evitare l&#39;apoptosi indotta da E2 e consentire l&#39;avvio della cancerogenesi.

Nucleo-citoplasmatici spola di proteine Papillomavirus E2 umane ad alto rischio induce l&#39;apoptosi.

Human papillomavirus (HPV) life cycle requires extensive manipulation of cell signalling to provide conditions adequate for viral replication within the stratified epithelia. In this regard, we show that the E2 regulatory protein of Î±, Î² and Î¼-HPV genotypes enhances tumour necrosis factor (TNF)-induced activation of nuclear factor kappa B (NF-ÎºB). This activation is mediated by the N-terminal domain of E2, but does not rely on its transcriptional properties. It is independent of the NF-ÎºB regulator Tax1BP1, which nevertheless interacts with all the E2 proteins. E2 specifically activates NF-ÎºB pathways induced by TNF, while interleukin-1-induced pathways are not affected. E2 stimulates the activating K63-linked ubiquitination of TRAF5, and interacts with both TRAF5 and TRAF6. Our data suggest that E2 potentiates TNF-induced NF-ÎºB signalling mediated by TRAF5 activation through direct binding. Since NF-ÎºB controls epithelial differentiation, this activity may be involved in the commitment of infected keratinocytes to proliferation arrest and differentiation, both required for the implementation of the productive viral cycle.

Keratinocyte sensitization to tumour necrosis factor-induced nuclear factor kappa B activation by the E2 regulatory protein of human papillomaviruses.

Cycle de vie des virus du papillome humain (VPH) n&eacute;cessite une vaste manipulation de cellule signalisation assurer des conditions ad&eacute;quates pour la r&eacute;plication virale dans l&#39;&eacute;pith&eacute;lium stratifi&eacute;. &Agrave; cet &eacute;gard, nous montrons que la prot&eacute;ine r&eacute;gulatrice E2 de &alpha;, &beta; et &mu;-HPV de g&eacute;notypes am&eacute;liore le facteur de n&eacute;crose tumorale (TNF)-induite par l&#39;activation de kappa facteur nucl&eacute;aire B (NF-&kappa;B). Cette activation est m&eacute;di&eacute;e par le domaine N-terminal de E2, mais ne repose pas sur ses propri&eacute;t&eacute;s transcriptionnelles. Il est ind&eacute;pendant de l&#39;organisme de r&eacute;glementation de NF-&kappa;B Tax1BP1, qui n&eacute;anmoins interagit avec toutes les prot&eacute;ines E2. E2 active sp&eacute;cifiquement les voies de NF-&kappa;B induites par le TNF, tandis que les voies induite par interleukin-1 ne sont pas affect&eacute;s. E2 stimule l&#39;activation ubiquitination K63-li&eacute; de TRAF5 et interagit avec les TRAF5 et les TRAF6. Nos r&eacute;sultats sugg&egrave;rent que E2 l&#39;potentialise induite par le TNF NF-&kappa;B signalisation m&eacute;di&eacute; par l&#39;activation de TRAF5 via la liaison directe. Comme NF-&kappa;B contr&ocirc;le diff&eacute;renciation &eacute;pith&eacute;liale, cette activit&eacute; pourrait &ecirc;tre impliqu&eacute;e dans l&#39;engagement des k&eacute;ratinocytes infect&eacute;s &agrave; l&#39;arr&ecirc;t de la prolif&eacute;ration et la diff&eacute;renciation, toutes deux n&eacute;cessaires &agrave; l&#39;ex&eacute;cution du cycle viral productif.

Sensibilisation des k&eacute;ratinocytes &agrave; de tumeur n&eacute;crose facteur nucl&eacute;aire induite par le facteur kappa B activation de la prot&eacute;ine r&eacute;gulatrice E2 du virus du papillome humain.

Ciclo di vita di papillomavirus umano (HPV) richiede la vasta manipolazione della cella di segnalazione per fornire le condizioni adeguate per la replicazione virale all&#39;interno dell&#39;epitelio stratificato. A questo proposito, ci mostra che la proteina normativa E2 di &alpha;, &beta; e &mu;-HPV genotipi migliora il fattore di necrosi tumorale (TNF)-indotta da attivazione di kappa nucleare fattore B (NF-&kappa;B). Questa attivazione &egrave; mediata dal dominio N-terminale della E2, ma non fare affidamento sulle sue propriet&agrave; di transcriptional. &Egrave; indipendente del regolatore di NF-&kappa;B Tax1BP1, che tuttavia interagisce con tutte le proteine E2. E2 specificamente attiva percorsi di NF-&kappa;B indotte da TNF, mentre percorsi di interleuchina-1-indotta non sono interessati. E2 stimola l&#39;attivazione ubiquitination K63-collegato di TRAF5 e interagisce con TRAF5 e TRAF6. I nostri dati suggeriscono che E2 potenzia il NF-&kappa;B indotta da TNF segnalazione mediata da TRAF5 attivazione attraverso il binding diretto. Poich&eacute; NF-&kappa;B controlla la differenziazione epiteliale, questa attivit&agrave; pu&ograve; essere coinvolta nell&#39;impegno di cheratinociti infetti all&#39;arresto della proliferazione e differenziazione, necessaria per l&#39;implementazione del ciclo produttivo virale.

Sensibilizzazione dei cheratinociti a tumore necrosi indotta dal fattore fattore nucleare kappa B attivazione dalla proteina E2 normativa del papillomavirus umano.

Benchmarking a luciferase complementation assay for detecting protein complexes.

Analyse comparative d&#39;une compl&eacute;mentation de luciferase pour la d&eacute;tection des complexes prot&eacute;iques.

Analisi comparativa di un&#39;analisi di complementazione di luciferase per la rilevazione di complessi della proteina.

Human Papillomaviruses (HPV) cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV). To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes.

Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new insights for e2 molecular functions.

Virus du papillome humain (VPH) causent des infections tr&egrave;s r&eacute;pandues chez les humains, entra&icirc;nant des infections latentes ou maladies allant de l&#39;hyperplasie b&eacute;nigne de prostate aux cancers. Pathologies induites par les HPV r&eacute;sultent d&#39;interactions complexes entre les prot&eacute;ines virales et du prot&eacute;ome de l&#39;h&ocirc;te. Compte tenu de la pr&eacute;occupation de sant&eacute; publique majeur due aux cancers associ&eacute;es au VPH, la plupart des &eacute;tudes ont port&eacute; sur les d&eacute;but prot&eacute;ines exprim&eacute;es par les g&eacute;notypes de VPH &agrave; haut potentiel oncog&eacute;nique (d&eacute;sign&eacute; &agrave; haut risque de HPV ou VPH). Pour faire progresser la compr&eacute;hension globale de la pathogen&egrave;se de l&#39;HPV, nous avons cartographi&eacute; les r&eacute;seaux d&#39;interaction virus/h&ocirc;te de la prot&eacute;ine r&eacute;gulatrice E2 de 12 repr&eacute;sentant de g&eacute;notypes de la pathog&eacute;nicit&eacute; de la gamme de HPV. Identification &agrave; grande &eacute;chelle des partenaires E2-interaction a &eacute;t&eacute; r&eacute;alis&eacute;e par des projections de deux-hybride de levure d&#39;une banque d&#39;ADNc HaCaT. Bas&eacute; sur un sch&eacute;ma de score de haut-confiance, un sous-ensemble de ces partenaires a &eacute;t&eacute; ensuite valid&eacute; pour l&#39;interaction par paire dans les cellules de mammif&egrave;res avec toute la gamme des 12 prot&eacute;ines E2, ce qui permet une analyse comparative d&#39;interaction. Regroupement hi&eacute;rarchique d&#39;interaction h&ocirc;te-E2 profils surtout recapitulated phylog&eacute;nie HPV et fournit des indices sur l&#39;implication de E2 dans l&#39;infection &agrave; HPV. Un ensemble de prot&eacute;ines cellulaires puisse donc &ecirc;tre identifi&eacute; une discrimination, entre le VPH muqueux, E2 prot&eacute;ines du VPH 16 ou 18 du VPH g&eacute;nital non oncog&egrave;ne. L&#39;&eacute;tude des r&eacute;seaux d&#39;interactions r&eacute;v&egrave;le un d&eacute;tournement pr&eacute;f&eacute;rentiel des prot&eacute;ines cellulaires tr&egrave;s branch&eacute;s et le ciblage de plusieurs familles fonctionnelles. Il s&#39;agit de r&eacute;gulation de la transcription, r&eacute;gulation de l&#39;apoptose, ARN, ubiquitination et trafic intracellulaire. Ce travail donne un aper&ccedil;u des fonctions biologiques de E2 &agrave; travers plusieurs g&eacute;notypes de VPH.

Comparaison de g&eacute;notype &agrave; grande &eacute;chelle du virus du papillome humain r&eacute;seaux d&#39;interactions h&ocirc;te-E2 fournit des nouvelles perspectives pour e2 fonctions mol&eacute;culaires.

Papillomavirus umano (HPV) provoca infezioni diffuse negli esseri umani, causando infezioni latenti o malattie che vanno da iperplasia benigna a tumori. Patologie indotte da HPV risultano di giochi complessi tra proteine virali e il proteoma host. Data la preoccupazione di salute pubblica a causa di tumori HPV-associati, maggior parte degli studi si sono concentrati sulle prime proteine espresse da HPV genotipi con alto potenziale oncogenico (designato ad alto rischio di HPV o HR-HPV). Per far avanzare la comprensione globale della patogenesi dell&#39;HPV, abbiamo mappato le reti di interazione virus/ospite della proteina E2 normativa da 12 rappresentante di genotipi di patogenicit&agrave; gamma di HPV. Identificazione su larga scala dei partner E2-interazione &egrave; stata eseguita da proiezioni di due-ibrido del lievito di una libreria di cDNA HaCaT. Basato su uno schema di punteggio alto-fiducia, un sottoinsieme di questi partner &egrave; stato poi convalidato per interazione pairwise in cellule di mammiferi con l&#39;intera gamma delle proteine E2 12, consentendo un&#39;analisi comparativa di interazione. Clustering gerarchico dell&#39;interazione ospite-E2 profili principalmente recapitulated filogenesi HPV e fornisce indizi per il coinvolgimento di E2 nell&#39;infezione da HPV. Una serie di proteine cellulari potrebbe essere identificata cos&igrave; discriminante, tra l&#39;HPV mucosali, proteine E2 di HR-HPV 16 o 18 dal non-oncogenica HPV genitale. Lo studio delle reti di interazione ha rivelato un dirottamento preferenziale delle proteine cellulari altamente connessi e la destinazione delle diverse famiglie funzionali. Questi includono la regolazione di trascrizione, regolazione del apoptosis, RNA processing, ubiquitination e traffico intracellulare. Il presente lavoro fornisce una panoramica delle funzioni biologiche E2 attraverso molteplici genotipi HPV.

Confronto genotipo su larga scala di papillomavirus umano reti di interazione E2-host fornisce nuove intuizioni per e2 funzioni molecolari.

Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or "interologs". Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus-host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interaction assays: yeast two-hybrid (Y2H), and our recently described "high-throughput Gaussia princeps protein complementation assay" (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split G. princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus-host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism-host interactions.

Comparative analysis of virus-host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase.

Interactomique comparative est une strat&eacute;gie pour inf&eacute;rer les interactions potentielles entre les prot&eacute;ines orthologues ou &laquo; interologs &raquo;. Ici nous nous concentrons, contrairement &agrave; l&#39;inf&eacute;rence standard bas&eacute;e sur l&#39;homologie, sur la divergence des profils d&#39;interaction de prot&eacute;ines parmi les organismes apparent&eacute;s, montrant que l&#39;approche peut corr&eacute;ler des traits sp&eacute;cifiques aux diff&eacute;rences ph&eacute;notypiques. Comme un mod&egrave;le, cette nouvelle approche comparative interactomic a &eacute;t&eacute; appliqu&eacute;e &agrave; grande &eacute;chelle aux prot&eacute;ines de virus du papillome humain (HPV). Le potentiel oncog&egrave;ne du HPV est principalement d&eacute;termin&eacute; par les prot&eacute;ines pr&eacute;coces E6 et E7. Nous avons cartographi&eacute; et chevauchent les r&eacute;seaux d&#39;interactions prot&eacute;iques virus-h&ocirc;te de prot&eacute;ines E6 et E7 11 g&eacute;notypes de VPH distinctes, s&eacute;lectionn&eacute;s pour leurs diff&eacute;rents Tropismes et les pathologies. Nous avons g&eacute;n&eacute;r&eacute; des ensembles de donn&eacute;es robuste et complet en combinant deux essais d&#39;interaction de prot&eacute;ine orthogonale : levure-hybride (Y2H) et notre r&eacute;cemment d&eacute;crite &laquo; high-throughput Gaussia princeps prot&eacute;ine analyse de compl&eacute;mentation &raquo; (HT-ACPP). HT-ACPP d&eacute;tecte l&#39;interaction de la prot&eacute;ine en mesurant la reconstitution induite par l&#39;interaction de l&#39;activit&eacute; d&#39;un split g. princeps lucif&eacute;rase. Regroupement hi&eacute;rarchique des profils d&#39;interaction r&eacute;capitul&eacute; phylog&eacute;nie HPV et a &eacute;t&eacute; utilis&eacute; pour corr&eacute;ler les profils d&#39;interaction virus-h&ocirc;te sp&eacute;cifique avec des traits pathologiques, refl&eacute;tant les potentiels canc&eacute;rog&egrave;nes distinctes des diff&eacute;rents HPV. Ce comparatif interactomique constitue une strat&eacute;gie fiable et puissante pour d&eacute;chiffrer les relations mol&eacute;culaires dans pratiquement n&#39;importe quelle combinaison des interactions microorganismes-h&ocirc;te.

Analyse comparative d&#39;interactomes virus-h&ocirc;te avec un dosage de compl&eacute;mentation de mammif&egrave;res prot&eacute;ine haut d&eacute;bit bas&eacute;e sur Gaussia princeps lucif&eacute;rase.

Interattomica comparativa &egrave; una strategia per la deduzione di potenziali interazioni tra proteine orthologous o &quot;interologs&quot;. Qui ci concentriamo, in contrasto con standard inferenza basata su omologia, sulla divergenza dei profili di interazione della proteina tra organismi strettamente correlati, mostrando che l&#39;approccio pu&ograve; correlare specifici tratti di differenze fenotipiche. Come un modello, questo nuovo approccio comparativo interactomic &egrave; stato applicato a larga scala di proteine di papillomavirus umano (HPV). Il potenziale oncogenico del HPV &egrave; determinato principalmente dalle proteine E6 ed E7 precoce. Abbiamo mappato e le reti di interazione della proteina virus-ospite delle proteine E6 ed E7 da 11 diversi genotipi HPV, selezionati per la loro diverse tropismi e patologie si sovrappongono. Abbiamo generato robusto e completo set di dati dalla combinazione di due analisi di interazione proteina ortogonale: lievito due-ibrido (Y2H) e il nostro recentemente descritto &quot;high-throughput Gaussia princeps complementazione analisi della proteina&quot; (HT-GPCA). HT-GPCA rileva interazione proteina misurando la ricostituzione dell&#39;attivit&agrave; di un split g. princeps luciferase interazione mediata. Clustering gerarchico dei profili di interazione ricapitolate filogenesi HPV e fu utilizzato per correlare i profili di interazione virus-ospite specifico con tratti patologici, riflettendo i potenziali cancerogeni distinti di HPV diverso. Questo interattomica comparativa costituisce una strategia potente e affidabile per decifrare i rapporti molecolari in praticamente qualsiasi combinazione delle interazioni microrganismo-ospite.

Analisi comparativa dei virus-host interactomes con un dosaggio di complementazione dei mammiferi high-throughput di proteine basato su Gaussia princeps luciferasi.

Over 100 genotypes of human papillomaviruses (HPVs) have been identified as being responsible for unapparent infections or for lesions ranging from benign skin or genital warts to cancer. The pathogenesis of HPV results from complex relationships between viral and host factors, driven in particular by the interplay between the host proteome and the early viral proteins. The E2 protein regulates the transcription, the replication as well as the mitotic segregation of the viral genome through the recruitment of host cell factors to the HPV regulatory region. It is thereby a pivotal factor for the productive viral life cycle and for viral persistence, a major risk factor for cancer development. In addition, the E2 proteins have been shown to engage numerous interactions through which they play important roles in modulating the host cell. Such E2 activities are probably contributing to create cell conditions appropriate for the successive stages of the viral life cycle, and some of these activities have been demonstrated only for the oncogenic high-risk HPV. The recent mapping of E2-host protein-protein interactions with 12 genotypes representative of HPV diversity has shed some light on the large complexity of the host cell hijacking and on its diversity according to viral genotypes. This article reviews the functions of E2 as they emerge from the E2/host proteome interplay, taking into account the large-scale comparative interactomic study.

The HPV E2-Host Protein-Protein Interactions: A Complex Hijacking of the Cellular Network.

Plus de 100 g&eacute;notypes du virus du papillome humain (HPV) ont &eacute;t&eacute; identifi&eacute;s comme &eacute;tant responsable des infections inapparente ou de l&eacute;sions allant de b&eacute;nignes de la peau ou des verrues g&eacute;nitales au cancer. La pathog&eacute;nie du VPH r&eacute;sultant des relations complexes entre viral and host factors, pilot&eacute;es notamment par l&#39;interaction entre le prot&eacute;ome de l&#39;h&ocirc;te et les prot&eacute;ines virales pr&eacute;coces. La prot&eacute;ine E2 r&eacute;gule la transcription, la reproduction ainsi que la s&eacute;gr&eacute;gation mitotique du g&eacute;nome viral par le recrutement des facteurs cellule h&ocirc;te &agrave; la r&eacute;gion r&eacute;gulatrice de HPV. C&#39;est donc un facteur crucial pour le cycle de vie viral productif et &agrave; la persistance virale, un facteur de risque majeur pour le d&eacute;veloppement du cancer. En outre, les prot&eacute;ines E2 montrent &agrave; s&#39;engager de nombreuses interactions &agrave; travers lequel ils jouent un r&ocirc;le important dans la modulation de la cellule h&ocirc;te. Ces activit&eacute;s de E2 contribuent probablement pour cr&eacute;er les conditions de la cellule appropri&eacute;e pour les &eacute;tapes successives du cycle de vie viral, et certaines de ces activit&eacute;s ont &eacute;t&eacute; d&eacute;montr&eacute;s que pour le HPV &agrave; haut risque oncog&egrave;ne. La cartographie r&eacute;cente d&#39;interactions prot&eacute;ine-prot&eacute;ine E2-h&ocirc;te avec 12 g&eacute;notypes repr&eacute;sentatifs de la diversit&eacute; de l&#39;HPV a faire la lumi&egrave;re sur la grande complexit&eacute; du d&eacute;tournement de cellule h&ocirc;te et sur sa diversit&eacute; selon les g&eacute;notypes viraux. Cet article passe en revue les fonctions de E2 tels qu&#39;ils ressortent de l&#39;interaction du prot&eacute;ome E2/h&ocirc;te, en tenant compte de l&#39;&eacute;tude &agrave; grande &eacute;chelle interactomic comparative.

Les Interactions de prot&eacute;ine-prot&eacute;ine E2-h&ocirc;te contre le VPH : Un d&eacute;tournement Complex du r&eacute;seau cellulaire.

Pi&ugrave; di 100 genotipi di papillomavirus umano (HPV) sono state identificate come responsabili assenti infezioni o lesioni che vanno dalla pelle benigna o verruche genitali al cancro. La patogenesi dell&#39;HPV deriva da complesse relazioni tra virale e ospitare fattori, guidate in particolare dall&#39;interazione tra il proteoma host e le prime proteine virali. La proteina E2 regola la trascrizione, la replica, nonch&eacute; la segregazione mitotica del genoma virale attraverso l&#39;assunzione di fattori cellula ospite alla regione normativa HPV. &Egrave; quindi un fattore cruciale per il ciclo di vita virale produttivo e per la persistenza virale, un importante fattore di rischio per lo sviluppo del cancro. Inoltre, le proteine E2 hanno dimostrate di impegnarsi numerose interazioni attraverso cui giocano ruoli importanti nel modulare la cellula ospite. Tali attivit&agrave; E2 sono probabilmente contribuendo a creare condizioni di cella appropriata per le fasi successive del ciclo di vita virale, e alcune di queste attivit&agrave; sono state dimostrate solo per l&#39;HPV ad alto rischio oncogenico. La recente mappatura delle interazioni della proteina-proteina E2-host con 12 genotipi rappresentativi della diversit&agrave; HPV ha fatto qualche luce sulla grande complessit&agrave; del dirottamento della cellula ospite e sulla sua diversit&agrave; secondo genotipi virali. Questo articolo recensisce le funzioni di E2 che emergono dall&#39;interazione proteome E2/host, prendendo in considerazione lo studio su grande scala comparativa interactomic.

Article	Year
Expression of ISWI and its binding to chromatin during the cell cycle and early development. Journal of structural biology\| PubMed ID: 12490154	2002
Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein. Oncogene\| PubMed ID: 12527886	2003
A genomic approach reveals a novel mitotic pathway in papillomavirus carcinogenesis. Cancer research\| PubMed ID: 14871818	2004
Nucleo-cytoplasmic shuttling of high risk human Papillomavirus E2 proteins induces apoptosis. The Journal of biological chemistry\| PubMed ID: 16135518	2005
Keratinocyte sensitization to tumour necrosis factor-induced nuclear factor kappa B activation by the E2 regulatory protein of human papillomaviruses. The Journal of general virology\| PubMed ID: 21715600	2011
Benchmarking a luciferase complementation assay for detecting protein complexes. Nature methods\| PubMed ID: 22127214	2011
Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new insights for e2 molecular functions. PLoS pathogens\| PubMed ID: 22761572	2012
Comparative analysis of virus-host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase. Methods (San Diego, Calif.)\| PubMed ID: 22898364	2012
The HPV E2-Host Protein-Protein Interactions: A Complex Hijacking of the Cellular Network. The open virology journal\| PubMed ID: 23341853	2012

Caroline Demeret

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