Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-14Bitstream added on 2014-06-13T18:31:21Z : No. of bitstreams: 1 yamasaki_lht_me_sjrp.pdf: 6194517 bytes, checksum: 350d37acd1e46ef2885eecc1354a317c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A infecção pelo vírus da Hepatite C (HCV) é considerada um grande problema de saúde pública, desde a sua descoberta em 1989. Entretanto a terapia mais utilizada atualmente, baseada no uso de Peginterferon, tem sucesso em aproximadamente 50% dos pacientes com o genótipo 1. Embora os mecanismos envolvidos nesta resistência viral ainda não sejam esclarecidos, sugere-se que fatores virais e do hospedeiro participam deste. A proteína não-estrutural 5A (NS5A) está envolvida em diversos processos celulares e é um componente essencial para o HCV. Entretanto, sua estrutura e função ainda não foram bem elucidadas. A partir destes fatos, os objetivos do presente estudo foram elaborar um modelo teórico da NS5A e investigar as propriedades estruturais e funcionais in silico. Foram analisadas 345 sequências da proteína NS5A do HCV de 23 pacientes infectados com o genótipo 1 ou 3. As composições de aminoácidos e de estrutura secundária demonstraram que há diferença entre os genótipos, podendo indicar que há diferenças nas interações proteína-proteína entre os genótipos, o que pode estar relacionado com a diferença da taxa de resistência ao tratamento. A análise funcional foi realizada com o ProtFun, que sugeriu que a NS5A estaria envolvida nas funções celulares de metabolismo intermediário central, tradução, crescimento, tranporte, ligação e hormônio. Estas funções variaram entre os domínios, suportando a hipótese de que a NS5A é uma proteína multifuncional. A análise pelo PROSITE indicou vários sítios de glicosilação, fosforilação e miristoilação, que são altamente conservados e podem ter função importante na estabilização da estrutura e função, sendo assim possíveis alvos de novos antivirais. Alguns deles estão em regiões relacionadas com a resposta ao tratamento. Outro... / Hepatitis C virus (HCV) infects almost 3% of people worldwide and it is considered the main cause of liver chronic diseases and transplants. Until today, there is no effective vaccine and the current most used therapy, based on Peginterferon, is successful only in 50% of patients infected by genotype 1. Although the outcomes of this treatment resistance are unclear, it is suggested host and virus factors may participate in this mechanism. Non-structural 5A (NS5A) protein is involved in several cellular and virus processes and it is a critical component of HCV. However, its structure and function are still uncertain. Regarding these facts, the present study attachments were to elaborate a model of the NS5A protein and to investigate NS5A structural and functional features, using in silico tools. It was analyzed 345 sequences of HCV NS5A protein from 23 patients infected by genotypes 1 or 3. Residues and secondary structure composition of all sequences demonstrated that there are differences between genotypes. It may indicate that there are differences in interactions between genotypes, which could be related with the distinct average of treatment resistance. In addition, among those that varied between genotypes, there were amino acids in regions that studies suggested as related with virus persistence. Functional analysis was performed with ProtFun. It suggested that NS5A is involved with central intermediary metabolism, translation, growth, transport, ligation and hormone functions in the cell. These functions vary between the domains, strengthening the hypothesis that NS5A is a multifunctional protein. Prosite motif search indicated that there are many glicosilation, fosforilation and myristoilation sites, which are highly conserved and may play an important role in structural stabilization and... (Complete abstract click electronic access below)

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

La protéine NS5A est essentielle pour la réplication et l'assemblage du virus de l'hépatite C (VHC), et elle constitue une cible thérapeutique prometteuse pour le développement d'antiviraux. Cependant, aucune fonction claire n'a encore été décrite pour NS5A, et les connaissances structurales restent limitées. Ainsi, nous avons caractérisé l'état intrinsèquement désordonné des domaines D2 et D3 de NS5A en décrivant leurs espaces conformationnels et leurs potentialités de repliement en combinant différentes méthodes biophysiques. Nous avons aussi mis en évidence la variabilité structurale du domaine D2 au sein des génotypes du VHC, ce qui pourrait être en rapport avec les différences de pathogénie et d'efficacité des thérapies observées selon les génotypes. L'interaction de D2 et D3 avec la cyclophiline humaine A (CypA) a été étudiée par résonance plasmonique de surface (SPR). Bien que des mutations au sein du domaine D2 rendent la réplication du VHC moins dépendante de la présence de CypA, ces mutations n'empêchent pas la liaison entre D2 et CypA. En revanche, elles induisent des perturbations structurales qui pourraient affecter la cinétique d'interconversion des conformères de D2. Nous avons montré par SPR que D2 et D3 interagissent avec le domaine de fixation à l'ADN du récepteur nucléaire FXR. Cette interaction pourrait inhiber la fixation de FXR sur sa cible ADN, suggérant une implication de NS5A dans la modulation de l'activité transcriptionnelle de ce récepteur nucléaire. L'ensemble de ces informations, nous a permis de proposer un modèle de la structure globale de NS5A permettant une meilleure compréhension des propriétés structurales et fonctionnelles de cette protéine énigmatique / NS5A is essential for HCV replication and particle assembly, and constitutes a very promising drug target. However, no clear function has yet been described for NS5A, and structural knowledge remains limited. We characterized the intrinsically disordered nature of NS5A domains D2 and D3, and describe their folding propensity and their overall conformational behaviour by combining different biophysical methods. We also highlighted the structural variability of D2 domain in HCV genotypes, which might be correlated with the disparities observed between genotypes in terms of pathogenesis and efficiency of therapies. The interactions between D2 and D3 with human cyclophilin A (CypA) was analysed by surface plasmon resonance (SPR). We showed that mutations in the D2 domain conferring resistance of HCV replication to CypA inhibitors did not prevent the interaction between D2 and CypA. However, they induce structural perturbations that may affect the kinetics of conformers interconversion of D2. We also showed by SPR that D2 and D3 interact with the of DNA-binding domain of the nuclear receptor FXR (farnesoid X receptor alpha). This interaction reduce the binding of FXR to its DNA target, suggesting an involvement of NS5A in the modulation of the transcriptional activity of FXR. All this data led us to propose a model of the overall structure of NS5A, which provides a useful template for a better understanding of structural and functional properties of this enigmatic protein