O Vírus Respiratório Sincicial Humano, do inglês human Respiratory Syncytial Virus (hRSV), é uma das maiores causas de doenças respiratórias agudas, principalmente em crianças e bebês entre seis meses e dois anos de idade. Não há drogas eficazes ou vacina aprovada até o momento para esse vírus, apesar das décadas de intensa pesquisa e grande quantidade de dados sobre ele acumulados. O genoma do hRSV codifica onze proteínas e a compreensão das interações entre essas proteínas virais e as proteínas do hospedeiro é essencial para que possíveis alvos terapêuticos contra o hRSV sejam identificados. No laboratório, anteriormente, foi dado enfoque às interações entre as proteínas celulares e as proteínas virais de matriz (M), nucleoproteína (N) e fosfoproteína (P). Neste trabalho, analisamos as interações da proteína viral M2-1 (cofator essencial para a transcrição) através da mesma estratégia utilizada naqueles experimentos, de fusão a FLAG (gerando FLAG-M2-1) e imunoprecipitação com anticorpos contra esse peptídeo. As proteínas co-imunoprecipitadas, identificadas por espectrometria de massas, foram: poly(A)-binding protein cytoplasmic 1 (PABPC1), Y-box binding protein 3 (YBX3), e Nuclease-sensitive element-binding protein 1 (YBX1). M2-1 é capaz de integrar-se ao complexo chamado de semelhante a corpúsculos de inclusão (IB like, do inglês), formado por N e P, que é similar estruturalmente aos corpúsculos de inclusão encontrados em células infectadas (IBs). Essa propriedade foi usada para analisar que proteínas celulares seriam recrutadas para esse outro nível de organização dessas três proteínas virais, envolvidas na transcrição. O complexo FLAG-N/P/M2-1 co-imunoprecipitou as proteínas celulares: Hsp70, Hsp90 (Heat shock proteins 70 e 90), Npm (Nucleophosmin), que podemos agrupar como chaperonas; PABPC1, YBX1, YBX3, ligantes de RNA; e sub-unidade pICIn do metilossomo, associada a modificação pós-tradução. Detalhamos a análise para YBX3, obtendo evidências adicionais de sua interação com M2-1 em ensaios de complementação de proteína fragmentada (Split-NanoLuc), e de co-localização por imunofluorescência indireta. Finalmente, utilizamos a metodologia de expressão em bactérias para demonstrar a interação entre M2-1 e os domínios funcionais de PABPC1, porém esses ensaios não foram conclusivos. / Human Respiratory Syncytial Virus (hRSV) is one of the leading causes of acute respiratory diseases, especially in children and infants between six months and two years of age. There is no effective drug or vaccine approved so far for this virus, despite decades of intensive research and large amount of data on it. The genome of hRSV encodes 11 proteins and the understanding of the interactions between these viral proteins and host proteins is essential to identify possible therapeutic targets against hRSV. In the lab, previously, was given focus to the interactions between cellular proteins and viral proteins matrix (M), nucleoprotein (N) and phosphoprotein (P). In this paper, we analyze the viral M2-1 (cofactor essential for transcription) protein interactions through the same strategy used in those experiments: fusion with FLAG (generating FLAG-M2-1) and immunoprecipitation with antibodies against this peptide. The co-immunoprecipitated proteins, identified by mass spectrometry, were: Poly (A)-binding protein cytoplasmic 1 (PABPC1), Y-box binding protein 3 (YBX3), and Nuclease-sensitive element-binding protein 1 (YBX1). M2-1 is able to integrate the complex called similar to inclusion bodies (IB like), formed by N and P, which is similar structurally to the inclusion bodies found in infected cells (IBs). This property has been used to analyze which cellular proteins would be recruited for this new level of organization of these three viral proteins involved in transcription. The cellular proteins co-immunoprecipitated with the complex FLAG-N/P/M2-1, were: Hsp70, Hsp90 (Heat shock proteins 70 and 90), Npm (Nucleophosmin), that we can group as chaperones; PABPC1, YBX1, YBX3, RNA ligands; and the methylosome sub-unit pICIn, post-translational modification-associated. We detailed the analysis for YBX3, obtaining additional evidence of its interaction with M2-1 in fragmented protein complementation tests (Split-NanoLuc), and co-localization by indirect immunofluorescence. Finally, we used the methodology of expression in bacteria to demonstrate the interaction between M2-1 and functional domains of PABPC1, but these tests were not conclusive.

Le virus respiratoire syncytial (VRS) est un membre de la famille des Paramyxoviridae, des virus simple brin d'ARN non segmentés de polarité négative. Le virus respiratoire syncytial humain (VRSh) est la principale cause des maladies respiratoires chez les enfants, et il est la priorité des cibles vaccinales. Notre objectif est d'obtenir des réponses sur la structure et la dynamique des différents composants du complexe ARN polymérase ARN dépendante du VRS (RdRp) et sur leurs interactions, en utilisant la résonance magnétique nucléaire (RMN), en espérant pouvoir proposer des molécules qui inhibent ou perturbent ces interactions afin de développer des médicaments antiviraux spécifiques. Mon travail porte sur deux protéines du complexe RdRp: la phosphoprotéine P, qui est le co-facteur principal de la polymérase et elle est nécessaire à la fois pour la transcription virale et pour la réplication, et M2-1 qui est un facteur d'antiterminaison de la transcription. Notre but est de caractériser la structure de P, par rapport à d'autres protéines P des Mononegavirales, la P du VRSh est assez courte et ne comporte pas de domaines avec structure tertiaire stable en dehors du domaine de tétramérisation central résistant à la trypsine. L'analyse de séquence de P prédit la présence d'un domaine d'oligomérisation et de grandes extensions en N-et C-terminales intrinsèquement désordonnés. Cet agencement de domaine est confirmé par RMN. En outre, nous avons utilisé l'analyse de déplacements chimiques secondaires et des mesures de relaxation nucléaire en azote 15 pour montrer que des hélices transitoires sont formées dans les extrémités N- et C-terminales de P. A l'extrémité C-terminale, des hélices presque complètement formées semblent prolonger le domaine d'oligomérisation. A l'extrémité N-terminale des hélices transitoires formées coïncident avec les sites de liaison pour la nucléoprotéine du VRS et pour la liaison au co-facteur de transcription M2-1, comme montré par des expériences d'interaction par RMN. La protéine M2-1 du VRSh est un cofacteur du complexe RdRp essentiel pour la transcription virale en augmentant la processivité de la polymérase. M2-1 est une protéine modulaire qui se lie à l'ARN et interagit également avec la phosphoprotéine virale P. Ces propriétés de liaison sont liées au domaine globulaire de M2-1. Puisque La structure du domaine globulaire de M2-1 a été déjà résolue par mon équipe par RMN, donc nous avons pu montrer le chevauchement partiel des surfaces d'interaction de l'ARN et de P, sur le fragment monomère de M2-1 (58-177) par RMN, confirmant que cette protéine se lie à la phosphoprotéine P et à l'ARN de manière compétitive. Tous les résultats de RMN sont toujours confirmés par des tests fonctionnels par nos collaborateurs à l'INRA, Jouy-en-Josas. / Respiratory syncytial virus (RSV) is a member of the Paramyxoviridae family of non segmented, negative sense singlestranded RNA viruses. Human respiratory syncytial virus (hRSV) is major cause of respiratory diseases in children, and is prioritized vaccine targets. Our aim is to get answers about the structure and dynamics of different components of the RSV RNA dependent RNA polymerase complex (RdRp) and about their interactions, by using Nuclear Magnetic Resonance, as a prerequisite to rational drug design. My work focuses on two RSV proteins: the phosphoprotein, which is the main polymerase co-factor and necessary for both viral transcription and replication, and M2-1 which is the antitermination factor of transcription. Our aim is to characterize the structure of P. As compared to other P proteins of Mononegavirales, hRSV P is rather short and does not comprise domains with stable tertiary fold outside the central trypsin-resistant tetramerization domain. Sequence analysis of P predicts the presence of a helical oligomerization domain and large disordered N-and C-terminal extensions. This domain arrangement is confirmed by NMR. Moreover we used backbone chemical shift analysis and 15 N relaxation experiments to show that transient helices are formed in the N- and C-termini of P. At the C-terminus, nearly completely formed helices seem to prolong the oligomerization domain. At the N-terminus transiently formed helices coincide with the binding sites for the RSV nucleoprotein and for the transcription co-factor M2-1, as shown by NMR interaction experiments. The M2-1 protein of hRSV functions as an essential transcriptional cofactor of the viral (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P. These binding properties are related to the core region of M2-1. After solving the structure of the corresponding domain, we showed that partial overlap of the RNA and P interaction surfaces, determined by NMR on the monomeric M2-1(58-177) fragment, accounts for the previously observed competitive behavior of RNA versus P in M2-1 binding. The NMR results are always confirmed by functional tests by our collaborators at INRA, Jouy-en-Josas

Le Virus Respiratoire Syncytial (VRS) est le principal agent responsable d’infections respiratoires sévères chez les nourrissons et les veaux. Le génome du VRS est constitué d’un ARN simple brin de polarité négative qui est répliqué et transcrit par le complexe ARN-polymérase viral (RdRp). Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur anti-terminateur de transcription M2-1. Le but de ce travail était de mieux caractériser la structure et le fonctionnement de deux protéines du complexe RdRp: P et M2-1.M2-1 est un tétramère constitué de 4 domaines : un « doigt de zinc », un domaine d’oligomérisation hélicoïdal, une région flexible, un domaine globulaire interagissant avec l'ARN et P, et une région C-terminale désordonnée. À partir de la structure cristalline de M2-1 pleine longueur, j'ai identifié des résidus critiques sur le doigt de zinc et la région flexible pour l'activité d'anti-terminaison de transcription de M2-1.Par la suite j'ai identifié une région de P critique pour l’interaction P - M2-1 et montre qu’elle est nécessaire au recrutement de M2-1 dans des corps d’inclusion cytoplasmiques. Je montre également que la déphosphorylation de M2-1, nécessaire à la transcription virale, est modulée par un complexe formé entre P et la phosphatase cellulaire PP1.Enfin, la cyclopamine, composé chimique naturel, inhibe la réplication du VRS. Je démontre qu'une seule mutation R151K sur M2-1 est suffisante pour conférer une résistance virale à la cyclopamine. Ces données ouvrent de nouvelles perspectives pour le développement de futures thérapies contre le VRS. / Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants and calves. The RSV genome consists of a single strand, negative-sense RNA, which is replicated and transcribed by the viral RNA-dependent RNA polymerase complex (RdRp). This complex is composed of the nucleoprotein N, the large protein L, the phosphoprotein P and the transcription anti-terminator M2-1. The aim of this work was to better characterize the structure and function of P and M2-1.M2-1 is a tetramer with 4 domains: a zinc-finger, a helical oligomerization domain, a flexible region, a RNA and P binding core domain and a C-terminal disordered region. Based on the crystal structure of the full-length M2-1 protein, I identified residues in the zinc-finger and the flexible loop critical for M2-1 antitermination activity.Then I identified a region of P critical for P – M2-1 interaction and show that it is required for the recruitment of M2-1 to cytoplasmic inclusion bodies. I also show that M2-1 dephosphorylation, which is critical for viral transcription, is modulated by a complex formed by P and the cellular phosphatase protein-1 (PP1).Finally cyclopamine, a natural chemical compound, inhibits the RSV replication. I show that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine. These data open a new avenue for the development of future therapies against RSV infection.