Recentemente, uma nova molécula de sinalização bacteriana, o AMP dimérico cíclico (c-di-AMP) emergiu como um regulador central dos processos fisiológicos essenciais, tais como a homeostase celular, verificação da integridade do DNA e virulência bacteriana, entre outros. O c-di-AMP é produzido a partir da condensação de duas moléculas de adenosina trifosfato (ATP) por proteínas denominadas diadenilato ciclases, que contém o domínio DisA_N, também denominado DAC. Existem 2842 sequências de proteínas que contém o domínio DAC, provenientes de 2386 organismos encontradas no banco de dados Protein Families Database (Pfam). Essas proteínas são divididas em subfamílias sendo as três subfamílias mais abundantes: DacA (69,1%), proteínas de membrana associadas a sinalização intracelular de alterações decorrentes do meio externo; DisA (24,1%), primeira diadenilato ciclase a ser amplamente estudada, é uma proteína intracelular encontrada na forma de octâmeros ativos em solução, a qual, indiretamente, controla a divisão celular através da verificação da integridade do DNA e DacB (5,5%), proteínas citoplasmáticas expressa, particularmente, durante a formação de esporos bacterianos. Uma característica interessante é que a maioria dos organismos contém uma única e essencial proteína com domínio DAC. Os organismos que contém duas ou mais proteínas-DAC, tais como Clostridium e Bacillus spp., são uma exceção. Em Staphylococcus aureus (S. aureus), um patógeno humano oportunista e responsável por inúmeras doenças infecciosas, uma única diadenilato ciclase é encontrada pendurada na porção interna da membrana celular (Sau_DacA). A atividade desta proteína é potencialmente regulada através da interação direta com uma proteína YbbR-like, que contém um domínio sensor extracelular. Sau_DacA conserva todos os elementos-chave de uma diadenilato ciclase bacteriana, e por ser a única presente em S. aureus, revela-se um excelente alvo de estudo para o desenvolvimento de novos fins terapêuticos. No entanto, até o presente momento, existem poucas informações em relação a estrutura proteica, ao mecanismo de síntese de c-di-AMP e regulação do mecanismo de síntese de nucleotídeo destas proteínas, sendo, portanto, neste aspectos que o presente trabalho pretendeu contribuir. Através de uma série de ensaios, estruturais, calorimétricos, espectroscópicos e bioquímicos, aliados a mutações sítio-dirigidas, identificou-se a relevância de uma conformação dimérica para a estabilidade conformacional e térmica para a proteína ser funcionalmente ativa, assim como a importância dos motivos conservados DGA (Aspartato-Glicina-Alanina) e RHR (Arginina-Histidina-Arginina) para a atividade da Sau_DacA. O loop L5 localizado entre o sítio ativo e a interface dimérica mostrou-se relevante, uma vez que nele é encontrado o motivo DGA - de ligação ao ATP - e o mesmo encontra-se estabilizado em uma posição favorável para ligação do ATP, apenas na conformação dimérica da proteína. Nossos resultados aliados a dados da literatura possibilitaram a proposição de um mecanismo de síntese de c-di-AMP que deve ocorrer via encontro face-a-face de dois sítios de ligação de ATP presentes em dímeros proteicos distintos, podendo a taxa de síntese de o nucleotídeo sofrer interferência via interação proteína-proteína com a proteína receptora de sinal Sau_YbbR. Desta forma, contribuímos para uma melhor compreensão da estrutura e função da Sau_DacA, possibilitando o uso desta como alvo para o desenvolvimento de novos fármacos, uma vez que é sabido que a biossíntese de c-di-AMP é essencial para a maioria dos patógenos que o sintetizam. / Recently, a new bacterial signaling molecule, the dimeric cyclic AMP (c-di-AMP) has emerged as a central regulator of essential physiological processes, such as cell wall homeostasis, DNA integrity and bacterial virulence, among others. C-di-AMP is synthesized from two molecules of adenosine triphosphate (ATP) by proteins containing DisA_N domain, also called diadenilato cyclases (DACs). A survey in the Protein Families Database database (Pfam) found 2842 protein sequences containing the DAC domain, from 2386 different organisms. These proteins are divided into subfamilies and the three most abundant are: DacA (69,1%), a membrane protein associated with intracellular signaling resulting from an external environment change; DisA (24,1%), the first and most widely studied diadenilate cyclase, an intracellular protein found as active octamers in solution which indirectly controls cell division by DNA integrity verification; and DacB (5,5%), a cytoplasmic proteins, particularly expressed during bacterial spores formation. An interesting feature is that most organisms contain just a single and essential DAC-protein. Organisms containing two or more DAC-containing proteins, such as Clostridium and Bacillus spp., are exceptions. In Staphylococcus aureus (S. aureus), an opportunistic human pathogen responsible for some life-threating diseases, there is a single membrane attached diadenilate cyclase, hanging in the inner portion of the cell membrane (Sau_DacA). The activity of this protein is potentially regulated through direct interaction with YbbR, which contains an extracellular sensor domain. Sau_DacA conserves all key elements of bacterial di-adenylate cyclase, and for being the only di-adenylate cyclase from S. aureus, proves to be an excellent study target for new therapeutic purposes. However, to date, there is a lack of information about structure, c-di-AMP synthesis mechanism and regulation of nucleotide synthesis by Sau_DacA. Therefore, in this context the present work aims to contribute. Through a series of structural, calorimetric, spectroscopic and biochemical assays combined with site-directed mutations, we solved the structure of a soluble construct of Sau_DacA and identified a dimeric interface relevance for the conformational and thermal stability to the protein. This dimer is functionally active and highlights the importance of conserved motifs DGA (Aspartate-Glycine-Alanine) and RHR (Arginine-Histidine-Arginine) for the activity of Sau_DacA. The L5 loop, located between the active site and the dimer interface where is allocated the ATP binding motif (DGA), is stabilized in a favorable position for ATP binding, just in protein dimeric conformation. Our results combined with literary allowed us infer the synthesis of c-di-AMP occurs by face-to-face encounter of two distinct ATP binding site and its rate of synthesis could be regulated through direct protein interaction with. In this way, we contribute to a better understanding of Sau_DacA structure and function, assisting in its use as a target for new drugs development since it is known the biosynthesis of c-di-AMP is essential for most pathogens that synthesize.

Recentemente, uma nova molécula de sinalização bacteriana, o AMP dimérico cíclico (c-di-AMP) emergiu como um regulador central dos processos fisiológicos essenciais, tais como a homeostase celular, verificação da integridade do DNA e virulência bacteriana, entre outros. O c-di-AMP é produzido a partir da condensação de duas moléculas de adenosina trifosfato (ATP) por proteínas denominadas diadenilato ciclases, que contém o domínio DisA_N, também denominado DAC. Existem 2842 sequências de proteínas que contém o domínio DAC, provenientes de 2386 organismos encontradas no banco de dados Protein Families Database (Pfam). Essas proteínas são divididas em subfamílias sendo as três subfamílias mais abundantes: DacA (69,1%), proteínas de membrana associadas a sinalização intracelular de alterações decorrentes do meio externo; DisA (24,1%), primeira diadenilato ciclase a ser amplamente estudada, é uma proteína intracelular encontrada na forma de octâmeros ativos em solução, a qual, indiretamente, controla a divisão celular através da verificação da integridade do DNA e DacB (5,5%), proteínas citoplasmáticas expressa, particularmente, durante a formação de esporos bacterianos. Uma característica interessante é que a maioria dos organismos contém uma única e essencial proteína com domínio DAC. Os organismos que contém duas ou mais proteínas-DAC, tais como Clostridium e Bacillus spp., são uma exceção. Em Staphylococcus aureus (S. aureus), um patógeno humano oportunista e responsável por inúmeras doenças infecciosas, uma única diadenilato ciclase é encontrada pendurada na porção interna da membrana celular (Sau_DacA). A atividade desta proteína é potencialmente regulada através da interação direta com uma proteína YbbR-like, que contém um domínio sensor extracelular. Sau_DacA conserva todos os elementos-chave de uma diadenilato ciclase bacteriana, e por ser a única presente em S. aureus, revela-se um excelente alvo de estudo para o desenvolvimento de novos fins terapêuticos. No entanto, até o presente momento, existem poucas informações em relação a estrutura proteica, ao mecanismo de síntese de c-di-AMP e regulação do mecanismo de síntese de nucleotídeo destas proteínas, sendo, portanto, neste aspectos que o presente trabalho pretendeu contribuir. Através de uma série de ensaios, estruturais, calorimétricos, espectroscópicos e bioquímicos, aliados a mutações sítio-dirigidas, identificou-se a relevância de uma conformação dimérica para a estabilidade conformacional e térmica para a proteína ser funcionalmente ativa, assim como a importância dos motivos conservados DGA (Aspartato-Glicina-Alanina) e RHR (Arginina-Histidina-Arginina) para a atividade da Sau_DacA. O loop L5 localizado entre o sítio ativo e a interface dimérica mostrou-se relevante, uma vez que nele é encontrado o motivo DGA - de ligação ao ATP - e o mesmo encontra-se estabilizado em uma posição favorável para ligação do ATP, apenas na conformação dimérica da proteína. Nossos resultados aliados a dados da literatura possibilitaram a proposição de um mecanismo de síntese de c-di-AMP que deve ocorrer via encontro face-a-face de dois sítios de ligação de ATP presentes em dímeros proteicos distintos, podendo a taxa de síntese de o nucleotídeo sofrer interferência via interação proteína-proteína com a proteína receptora de sinal Sau_YbbR. Desta forma, contribuímos para uma melhor compreensão da estrutura e função da Sau_DacA, possibilitando o uso desta como alvo para o desenvolvimento de novos fármacos, uma vez que é sabido que a biossíntese de c-di-AMP é essencial para a maioria dos patógenos que o sintetizam. / Recently, a new bacterial signaling molecule, the dimeric cyclic AMP (c-di-AMP) has emerged as a central regulator of essential physiological processes, such as cell wall homeostasis, DNA integrity and bacterial virulence, among others. C-di-AMP is synthesized from two molecules of adenosine triphosphate (ATP) by proteins containing DisA_N domain, also called diadenilato cyclases (DACs). A survey in the Protein Families Database database (Pfam) found 2842 protein sequences containing the DAC domain, from 2386 different organisms. These proteins are divided into subfamilies and the three most abundant are: DacA (69,1%), a membrane protein associated with intracellular signaling resulting from an external environment change; DisA (24,1%), the first and most widely studied diadenilate cyclase, an intracellular protein found as active octamers in solution which indirectly controls cell division by DNA integrity verification; and DacB (5,5%), a cytoplasmic proteins, particularly expressed during bacterial spores formation. An interesting feature is that most organisms contain just a single and essential DAC-protein. Organisms containing two or more DAC-containing proteins, such as Clostridium and Bacillus spp., are exceptions. In Staphylococcus aureus (S. aureus), an opportunistic human pathogen responsible for some life-threating diseases, there is a single membrane attached diadenilate cyclase, hanging in the inner portion of the cell membrane (Sau_DacA). The activity of this protein is potentially regulated through direct interaction with YbbR, which contains an extracellular sensor domain. Sau_DacA conserves all key elements of bacterial di-adenylate cyclase, and for being the only di-adenylate cyclase from S. aureus, proves to be an excellent study target for new therapeutic purposes. However, to date, there is a lack of information about structure, c-di-AMP synthesis mechanism and regulation of nucleotide synthesis by Sau_DacA. Therefore, in this context the present work aims to contribute. Through a series of structural, calorimetric, spectroscopic and biochemical assays combined with site-directed mutations, we solved the structure of a soluble construct of Sau_DacA and identified a dimeric interface relevance for the conformational and thermal stability to the protein. This dimer is functionally active and highlights the importance of conserved motifs DGA (Aspartate-Glycine-Alanine) and RHR (Arginine-Histidine-Arginine) for the activity of Sau_DacA. The L5 loop, located between the active site and the dimer interface where is allocated the ATP binding motif (DGA), is stabilized in a favorable position for ATP binding, just in protein dimeric conformation. Our results combined with literary allowed us infer the synthesis of c-di-AMP occurs by face-to-face encounter of two distinct ATP binding site and its rate of synthesis could be regulated through direct protein interaction with. In this way, we contribute to a better understanding of Sau_DacA structure and function, assisting in its use as a target for new drugs development since it is known the biosynthesis of c-di-AMP is essential for most pathogens that synthesize.

Au cours du cycle réplicatif des rétrovirus, l’ADN viral rétro-transcrit est intégré dans l’ADN de la cellule hôte par l’intégrase virale (IN). L’IN possède un rôle clé dans le cycle rétroviral et représente une cible thérapeutique majeure pour le traitement des infections par le virus de l’immunodéficience humaine (VIH). L’IN est constituée de trois domaines (N-terminal, central et C-terminal) connectés par des boucles flexibles, qui la rendent difficilement cristallisable. Le Dr. C. Ronfort (Equipe Rétrovirus et Intégration Rétrovirale) et le Pr. P. Gouet (Laboratoire de BioCristallographie) collaborent depuis 2002 sur l’IN du Rous Associated Virus type 1 (RAV-1). Mes travaux de thèse s’inscrivent dans le cadre de cette collaboration. Il s’agissait de mener une étude cristallographique et moléculaire du domaine central de l’IN du RAV-1 pour pouvoir, ensuite, modéliser des mutants d’intérêt identifiés par l’équipe du Dr. C. Ronfort. Pour ce faire, le fragment protéique a été surproduit et purifié. Sa structure cristallographique a été résolue à une résolution de 1,8 Å. L’examen de cette structure révèle que le dimère de l’IN du RAV-1 peut s’assembler suivant une nouvelle interface moléculaire stabilisée par trois paires d’hélices α. Cet assemblage se caractérise également par la présence d’un étroit sillon basique à sa surface. Par des expériences in vitro de biochimie et in silico de docking, nous avons montré que ce sillon était susceptible de fixer un brin d’ARN. D’autre part, nos données expérimentales permettent d’expliquer comment les conditions de cristallisation, ainsi que la substitution d’un acide aminé de surface, favorisent la formation soit de ce nouvel arrangement dimérique, soit de l’arrangement dimérique classique. Ainsi, l’ensemble des données obtenues au cours de cette thèse suggère que l’intégrase possède des propriétés structurales modulables, lui permettant d’intervenir dans plusieurs étapes du cycle rétroviral en présence d’ADNdb (intégration) ou d’ARNsb (rétro-transcription et/ou encapsidation du génome ARN viral) / During the replicative cycle of retroviruses, the retrotranscribed viral DNA is integrated into the host chromosome by the viral integrase protein (IN). The integration reaction is essential for the viral life cycle. Therefore, IN is a key target for antiretroviral drug design to treat HIV infection. IN consists of three domains (N-terminal, central and Cterminal) connected by flexible loops, making the enzyme difficult to crystallize. Dr C. Ronfort (Team Retrovirus and Retroviral Integration) and Pr P. Gouet (BioCrystallography Laboratory) collaborate since 2002 in Lyon to study IN from the Rous Associated Virus type 1 (RAV-1). My thesis work lies within this collaboration. Its objective was to perform crystallographic and molecular studies of the central domain of RAV-1 IN and of mutants of interest identified by the team of Dr C. Ronfort. In this aim, the IN fragment has been overexpressed and purified. Its crystal structure has been solved to a resolution of 1.8 Å. The observation of this structure reveals that the RAV-1 IN can exhibit a novel dimeric arrangement with a molecular interface stabilized by three pairs of facing α-helices. This arrangement is also characterized by the presence of a basic narrow groove at its surface. Thanks to biochemical in vitro experiments and in silico docking studies, we have shown that this median groove could allow the binding of a linear singlestranded RNA. Moreover, our experimental data can explain how the crystallization conditions as well as the mutation of a specific residue located at the surface of the enzyme favor either this novel dimeric arrangement or the classical dimeric interface. Therefore, the data obtained during this thesis suggest that IN exhibits modular structural properties, allowing it to operate in several distinct steps of the retroviral cycle in presence of dsDNA (integration) or ssRNA (reverse transcription and/or encapsidation of the retroviral RNA genome)

Les papillomavirus sont de petits virus à ADN double brin qui infectent les cellules de l’épithélium de la peau et des muqueuses d’une variété de vertébrés causant des lésions bénignes telles des verrues. Certains de ces virus sont également associés au développement de lésions malignes, notamment le cancer du col utérin. La protéine régulatrice E2 des papillomavirus est impliquée dans diverses fonctions contribuant à l’établissement de l’infection par ces virus. Entre autre, E2 régule la transcription des gènes viraux, participe à l’initiation de la réplication de l’ADN viral en s’associant à l’hélicase virale E1 et est responsable du maintien et de la ségrégation de l’épisome viral au cours de la division cellulaire. Toutes ces activités sont attribuables à la capacité de E2 à s’associer au génome viral et à interagir avec des protéines virales et cellulaires. De plus, ces fonctions sont elles-mêmes régulées par des modifications post-traductionnelles de la protéine E2. Plusieurs études ont été réalisées afin de découvrir les mécanismes de régulation des fonctions de E2 mais le rôle exact des différents domaines de E2 dans ces contrôles reste à être défini. En premier lieu, nous nous sommes intéressés à l’interaction entre E2 et Brd4(L) qui avait été définie comme étant essentielle à la ségrégation de l’épisome. Plusieurs caractéristiques associées à la protéine Brd4(L) telles que sa capacité à lier les lysines acétylées des histones, son interaction avec le complexe Mediator et sa participation à l’activation de la transcription en formant un complexe avec pTEFb, nous ont permis d’émettre l’hypothèse que l’interaction E2-Brd4(L) est nécessaire à l’activité transcriptionnelle de E2. Nous avons démontré que la protéine Brd4(L) interagit avec le domaine de transactivation de E2 de divers types de papillomavirus. De plus, cette interaction implique les résidus de E2 essentiels à son activité transcriptionnelle. Ainsi, ces résultats proposent que l’association E2-Brd4(L) serve à la régulation de la transcription des gènes viraux. Dans un second temps, nos recherches se sont concentrées sur l’existence d’une interface de dimérisation au sein du domaine de transactivation de E2 et de son implication dans les activités transcriptionnelles et réplicatives de la protéine. Nos études ont aussi mis en évidence que l’intégrité de la structure de ce domaine contribue au bon fonctionnement de la réplication du génome viral. Cette découverte suggère que la dimérisation de E2 peut réguler l’initiation de la réplication et propose l’existence d’un niveau de régulation additionnel impliquant l’état de la structure quaternaire de la protéine E2 et une modulation de l’interaction entre E1 et E2 à cette étape du cycle viral. Finalement, l’étude de l’instabilité de la protéine E2 nous a permis de définir une région importante dans le domaine flexible de la protéine, nécessaire à sa dégradation par le protéasome. De plus, la présence de résidus conservés localisés dans ce domaine, sont associés à la dégradation et portent la signature d’un signal de localisation nucléaire de type PY-NLS, suggérant que la stabilité de la protéine E2 est régulée par sa localisation au sein de la cellule. Ces études démontrent l’existence de nouvelles stratégies de régulation des activités transcriptionnelle et réplicative de la protéine E2 des papillomavirus. La compréhension de ces mécanismes nous permet de mieux cerner les étapes favorisant l’établissement et la progression du cycle viral et d’identifier de nouvelles cibles thérapeutiques contre les infections aux papillomavirus. / Papillomaviridae is a family of small double-stranded DNA viruses known as papillomaviruses (PV) which infect skin and mucosal epithelial cells where they cause benign lesions such as warts. A subset of these viruses is associated with the development of malignant lesions and is the causal agent of cervical cancer. Papillomavirus E2 regulatory protein is involved in several functions leading to the establishment of the viral infection. These activities include the regulation of viral genes transcription, it participation to the initiation of viral DNA replication by recruiting the viral helicase E1, and to the maintenance and segregation of the viral episome during cellular division. All these functions are associated to the ability of E2 to bind specifically the viral genome, to interact with viral and cellular proteins and to acquire post-translational modifications. The first article of this thesis led to the identification of Brd4(L) as the major protein associated to E2 protein of different papillomavirus types. This interaction involves the amino acids associate to the transcription function of E2. The protein Brd4(L) was identified originally as a factor that maintains epigenetic memory by it interaction with acetylated histones during mitosis. This association with the chromatin, it interaction with Mediator complex and it participation to the cellular transcription by recruiting pTEFb complex allowed us to propose that the interaction between Brd4 and E2 is essential to the regulation of viral gene transcription. The second part of this work based on previous characterization of the transactivation domain dimerization interface; investigate the role of this surface in the transcriptional and replicative activities of E2. Our studies demonstrated that the integrity of the TAD dimerization interface may contribute to the DNA replication activity of E2. This discovery suggests that the dimerization interface may regulate the viral DNA replication by the redox state of the E2 protein. A fine characterization of this interface may provide new aspect of the interaction between E1 and E2 in the context of viral cycle. Finally, the third section of this thesis define a region of E2 protein associated to it degradation by the proteasome. This study also demonstrates that the stability of E2 is related to its cellular localization and suggests that the highly conserved residues found in this region may represent a PY-NLS nuclear localization signal signature. This thesis shows the existence of different approaches to regulate the transcriptional and the replicative activities as well as the stability of the papillomavirus E2 protein to favor the establishment and the progression of viral cycle.

Les papillomavirus sont de petits virus à ADN double brin qui infectent les cellules de l’épithélium de la peau et des muqueuses d’une variété de vertébrés causant des lésions bénignes telles des verrues. Certains de ces virus sont également associés au développement de lésions malignes, notamment le cancer du col utérin. La protéine régulatrice E2 des papillomavirus est impliquée dans diverses fonctions contribuant à l’établissement de l’infection par ces virus. Entre autre, E2 régule la transcription des gènes viraux, participe à l’initiation de la réplication de l’ADN viral en s’associant à l’hélicase virale E1 et est responsable du maintien et de la ségrégation de l’épisome viral au cours de la division cellulaire. Toutes ces activités sont attribuables à la capacité de E2 à s’associer au génome viral et à interagir avec des protéines virales et cellulaires. De plus, ces fonctions sont elles-mêmes régulées par des modifications post-traductionnelles de la protéine E2. Plusieurs études ont été réalisées afin de découvrir les mécanismes de régulation des fonctions de E2 mais le rôle exact des différents domaines de E2 dans ces contrôles reste à être défini. En premier lieu, nous nous sommes intéressés à l’interaction entre E2 et Brd4(L) qui avait été définie comme étant essentielle à la ségrégation de l’épisome. Plusieurs caractéristiques associées à la protéine Brd4(L) telles que sa capacité à lier les lysines acétylées des histones, son interaction avec le complexe Mediator et sa participation à l’activation de la transcription en formant un complexe avec pTEFb, nous ont permis d’émettre l’hypothèse que l’interaction E2-Brd4(L) est nécessaire à l’activité transcriptionnelle de E2. Nous avons démontré que la protéine Brd4(L) interagit avec le domaine de transactivation de E2 de divers types de papillomavirus. De plus, cette interaction implique les résidus de E2 essentiels à son activité transcriptionnelle. Ainsi, ces résultats proposent que l’association E2-Brd4(L) serve à la régulation de la transcription des gènes viraux. Dans un second temps, nos recherches se sont concentrées sur l’existence d’une interface de dimérisation au sein du domaine de transactivation de E2 et de son implication dans les activités transcriptionnelles et réplicatives de la protéine. Nos études ont aussi mis en évidence que l’intégrité de la structure de ce domaine contribue au bon fonctionnement de la réplication du génome viral. Cette découverte suggère que la dimérisation de E2 peut réguler l’initiation de la réplication et propose l’existence d’un niveau de régulation additionnel impliquant l’état de la structure quaternaire de la protéine E2 et une modulation de l’interaction entre E1 et E2 à cette étape du cycle viral. Finalement, l’étude de l’instabilité de la protéine E2 nous a permis de définir une région importante dans le domaine flexible de la protéine, nécessaire à sa dégradation par le protéasome. De plus, la présence de résidus conservés localisés dans ce domaine, sont associés à la dégradation et portent la signature d’un signal de localisation nucléaire de type PY-NLS, suggérant que la stabilité de la protéine E2 est régulée par sa localisation au sein de la cellule. Ces études démontrent l’existence de nouvelles stratégies de régulation des activités transcriptionnelle et réplicative de la protéine E2 des papillomavirus. La compréhension de ces mécanismes nous permet de mieux cerner les étapes favorisant l’établissement et la progression du cycle viral et d’identifier de nouvelles cibles thérapeutiques contre les infections aux papillomavirus. / Papillomaviridae is a family of small double-stranded DNA viruses known as papillomaviruses (PV) which infect skin and mucosal epithelial cells where they cause benign lesions such as warts. A subset of these viruses is associated with the development of malignant lesions and is the causal agent of cervical cancer. Papillomavirus E2 regulatory protein is involved in several functions leading to the establishment of the viral infection. These activities include the regulation of viral genes transcription, it participation to the initiation of viral DNA replication by recruiting the viral helicase E1, and to the maintenance and segregation of the viral episome during cellular division. All these functions are associated to the ability of E2 to bind specifically the viral genome, to interact with viral and cellular proteins and to acquire post-translational modifications. The first article of this thesis led to the identification of Brd4(L) as the major protein associated to E2 protein of different papillomavirus types. This interaction involves the amino acids associate to the transcription function of E2. The protein Brd4(L) was identified originally as a factor that maintains epigenetic memory by it interaction with acetylated histones during mitosis. This association with the chromatin, it interaction with Mediator complex and it participation to the cellular transcription by recruiting pTEFb complex allowed us to propose that the interaction between Brd4 and E2 is essential to the regulation of viral gene transcription. The second part of this work based on previous characterization of the transactivation domain dimerization interface; investigate the role of this surface in the transcriptional and replicative activities of E2. Our studies demonstrated that the integrity of the TAD dimerization interface may contribute to the DNA replication activity of E2. This discovery suggests that the dimerization interface may regulate the viral DNA replication by the redox state of the E2 protein. A fine characterization of this interface may provide new aspect of the interaction between E1 and E2 in the context of viral cycle. Finally, the third section of this thesis define a region of E2 protein associated to it degradation by the proteasome. This study also demonstrates that the stability of E2 is related to its cellular localization and suggests that the highly conserved residues found in this region may represent a PY-NLS nuclear localization signal signature. This thesis shows the existence of different approaches to regulate the transcriptional and the replicative activities as well as the stability of the papillomavirus E2 protein to favor the establishment and the progression of viral cycle.