A dengue é uma doença que afeta milhões de pessoas e possui um número significativo de mortes. Não há nenhum tratamento vacinal legalizado para uso. As estratégias vacinais contra a dengue baseadas em proteínas não estruturais têm demonstrado serem mais seguras do que as baseadas em proteínas estruturais. A proteína não estrutural 5 (NS5) do vírus dengue é a proteína mais conservada entre os quatro sorotipos e desempenha um papel crucial na replicação viral. Neste estudo, foi gerada uma forma recombinante da NS5 expressa em E. coli com propriedades antigênicas preservados. As condições de cultura foram optimizadas, o que permitiu a expressão dessa proteína na forma solúvel. A imunização de camundongos Balb/c com a NS5 sozinha ou em combinação com um adjuvante (poli (I:C)) promoveu o aumento da sobrevida de camundongos após desafio letal com DENV2. A combinação da NS5 com poli (I:C) emulsionado em Montanide 720 levou a expansão de linfócitos T CD8+ específicos. Os resultados indicam que a proteína NS5 obtida preserva determinantes antigênicos da proteína nativa e pode ser uma ferramenta útil para estudos sobre a biologia do DENV, busca de drogas antivirais e desenvolvimento de vacinas. / Dengue fever is a disease affecting millions of people worldwide and causing a significant number of deaths. There are no effective treatments or vaccine approaches capable of preventing such infection. Anti-DENV vaccine strategies based on nonstructural proteins as antigens have been shown to be safer than those based on structural proteins. The DENV nonstructural protein 5 (NS5), plays a crucial role in viral replication. In this study, we generated a recombinant form of DENV2 NS5 expressed in E. coli in high amounts and with preserved antigenic properties with regard to the native protein. Culture conditions were optimized in order to allow expression of NS5 as a soluble protein. The immunization of Balb/c mice using this protein alone or in combination with poly (I:C) led to increased survival after intracranial challenge with the DENV2 JHA1 strain. The combination of the protein with poly (I:C) emulsified in Montanide 720 led to the activation of NS5-specific CD8+ T lymphocytes. Altogether, the results indicate that the recombinant NS5 protein preserves antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV\'s biology, search for anti-viral drugs and vaccine development.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-15T11:01:10Z No. of bitstreams: 2 Dissertação - Tharlley Rodrigo Eugenio Duarte - 2018.pdf: 3238895 bytes, checksum: 4372ea2b66d739dd3a82a02b57e2b24a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-05-15T12:24:47Z (GMT) No. of bitstreams: 2 Dissertação - Tharlley Rodrigo Eugenio Duarte - 2018.pdf: 3238895 bytes, checksum: 4372ea2b66d739dd3a82a02b57e2b24a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-15T12:24:47Z (GMT). No. of bitstreams: 2 Dissertação - Tharlley Rodrigo Eugenio Duarte - 2018.pdf: 3238895 bytes, checksum: 4372ea2b66d739dd3a82a02b57e2b24a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-26 / Brazil is the largest arbovirus granary in the world and presents the largest endemic area of yellow fever (YF). The Ministry of Health reported in 1170 suspected cases of yellow fever, of which 847 are under investigation, 93 were discarded and 230 were confirmed, being in the states of Minas Gerais (201), Espírito Santo (25) and São Paulo (4). Of the total number of cases reported, 186 died, 104 of which remained in the investigation, 79 deaths were confirmed and 3 were discarded. The case fatality rate among confirmed cases was 34.3%. For YF there is no specific treatment, however, vaccination is effective being the only and best way of prevention. Precisely because of this factor and others involved the diagnosis of YF is not made in the health system except by a relevant suspicion. The problem is that in many cases viruses go unnoticed in cases of Dengue virus infection because of cross reactivity between members of the genus Flavívirus or because of non-specific symptoms. The present study analyzed 118 samples that were screened for suspected Dengue Virus infections (VDEN) for the year 2011 to 2013, but discarded for this virus because they gave negative results to the viral agent through serological and molecular tests in the municipality of Goiânia, Goiás. Samples were sent to the Virology Laboratory of the Federal University of Goiás Regional Jataí and analyzed by molecular methods such as RT-PCR and Nested-PCR followed by verification of the amplicon by agarose gel electrophoresis. Among the 118 negative samples for the DENV virus, three of the samples were positive for the Yellow Fever Virus (YVF) according to the production of amplicons of 253 bp of the NS5 region and confirmation of the identity of the amplicon by nucleotide sequencing. The sequences obtained were submitted to BLAST for identity confirmation. The translated sequence was analyzed by MEGA software version 7.0. For phylogenetic analysis, the best model was previously determined and then the tree was constructed with 53 sequences of all Yellow Fever viruses present in databases for a comparative analysis. The sequences found were compared to sequences from the VFA recorded in the Genbank database and were identified as referring to a portion of the NS5 nonstructural protein, position 216 to 296 of this protein, conferring 81 amino acids. The representative tree demonstrated that the sequences submitted were directly related to Senegal taxa. The robustness of the phylogenetic method was by Bootstrap 2000 replicates using the best JTT + G model, Maximum Likelihood. The Tajima test applied yielded a value of D = 1.159570, thus demonstrating that there was no population expansion of the taxa analyzed, considering that they have a significant degree of conservation during evolution. From the results obtained in the study, it can be affirmed that there was already an YFV circulation in the year 2013, at least of patients seen in the central region of Brazil, even before the last outbreak in 2017. / O Brasil é o maior celeiro de arbovírus do mundo e apresenta a maior área endêmica de febre amarela (FA). O Ministério da Saúde notificou, em 2017, 1170 casos suspeitos de febre amarela, sendo que desses 847 estão em investigação, 93 foram descartados e 230 foram confirmados, sendo nos estados de Minas Gerais (201), Espírito Santo (25) e São Paulo (4). Do total de casos notificados, 186 evoluíram para óbito, sendo que 104 óbitos permanecem em investigação, 79 óbitos foram confirmados e 3 foram descartados. A taxa de letalidade entre os casos confirmados foi de 34,3%. Para FA não há tratamento específico, no entanto, a vacinação é eficaz sendo a única e melhor maneira de prevenção. Justamente por esse fator e outros envolvidos o diagnóstico da FA não é feito no sistema de saúde a não ser por uma suspeita relevante. O problema é que em muitos casos os vírus passam despercebidos em casos de infecção pelo vírus Dengue por apresentar reatividade cruzada entre membros do gênero Flavívirus ou por apresentar sintomas inespecíficos. O presente estudo analisou 118 amostras que foram triadas para infecções suspeitas de Vírus da Dengue (VDEN) referentes ao ano de 2011 a 2013, porém descartadas para esta virose por terem dado resultados negativos para o agente viral através de testes sorológicos e moleculares no município de Goiânia, Goiás. As amostras foram encaminhadas para o Laboratório de Virologia da Universidade Federal de Goiás Regional Jataí e analisadas por métodos moleculares, como o de RT-PCR e Nested-PCR seguida da verificação do amplicon por eletroforese em gel de Agarose. Dentre as 118 amostras negativas para os vírus DENV, três das amostras foram positivas para o Vírus da Febre Amarela (VFA) conforme produção de amplicons de 253 pb da região NS5 e confirmação da identidade do amplicon por sequenciamento nucleotídico. As sequências obtidas foram submetidas ao BLAST para confirmação da identidade. A sequência traduzida foi analisada pelo software MEGA versão 7.0. Para análise filogenética foi determinado previamente o melhor modelo e em seguida a árvore foi construída com 53 sequências de todos os vírus da Febre Amarela presentes em bancos de dados para uma análise comparativa. As sequências encontradas foram comparadas com sequências do VFA registradas no banco de dados Genbank e identificouse que se refere a uma porção da proteína não estrutural NS5, posição 216 a 296 desta proteína, conferindo 81 aminoácidos. A árvore representativa demonstrou que as sequências submetidas estavam diretamente relacionadas com táxons do Senegal. A robustez do método filogenético foi por Bootstrap 2000 réplicas utilizando o melhor modelo JTT+G, Maximum Likelihood (Máxima probabilidade). O teste D de Tajima aplicado gerou um valor de D= 1.159570, demonstrando assim que não houve expansão populacional dos táxons analisados, considerando que os mesmos possuem significativo grau de conservação durante a evolução. A partir dos resultados obtidos no estudo, pode-se afirmar que já havia circulação do VFA no ano de 2013, pelo menos de pacientes atendidos na região central do Brasil, antes mesmo do último surto em 2017.

A dengue é uma doença que afeta milhões de pessoas e possui um número significativo de mortes. Não há nenhum tratamento vacinal legalizado para uso. As estratégias vacinais contra a dengue baseadas em proteínas não estruturais têm demonstrado serem mais seguras do que as baseadas em proteínas estruturais. A proteína não estrutural 5 (NS5) do vírus dengue é a proteína mais conservada entre os quatro sorotipos e desempenha um papel crucial na replicação viral. Neste estudo, foi gerada uma forma recombinante da NS5 expressa em E. coli com propriedades antigênicas preservados. As condições de cultura foram optimizadas, o que permitiu a expressão dessa proteína na forma solúvel. A imunização de camundongos Balb/c com a NS5 sozinha ou em combinação com um adjuvante (poli (I:C)) promoveu o aumento da sobrevida de camundongos após desafio letal com DENV2. A combinação da NS5 com poli (I:C) emulsionado em Montanide 720 levou a expansão de linfócitos T CD8+ específicos. Os resultados indicam que a proteína NS5 obtida preserva determinantes antigênicos da proteína nativa e pode ser uma ferramenta útil para estudos sobre a biologia do DENV, busca de drogas antivirais e desenvolvimento de vacinas. / Dengue fever is a disease affecting millions of people worldwide and causing a significant number of deaths. There are no effective treatments or vaccine approaches capable of preventing such infection. Anti-DENV vaccine strategies based on nonstructural proteins as antigens have been shown to be safer than those based on structural proteins. The DENV nonstructural protein 5 (NS5), plays a crucial role in viral replication. In this study, we generated a recombinant form of DENV2 NS5 expressed in E. coli in high amounts and with preserved antigenic properties with regard to the native protein. Culture conditions were optimized in order to allow expression of NS5 as a soluble protein. The immunization of Balb/c mice using this protein alone or in combination with poly (I:C) led to increased survival after intracranial challenge with the DENV2 JHA1 strain. The combination of the protein with poly (I:C) emulsified in Montanide 720 led to the activation of NS5-specific CD8+ T lymphocytes. Altogether, the results indicate that the recombinant NS5 protein preserves antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV\'s biology, search for anti-viral drugs and vaccine development.

La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypes. / Dengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes.

Les Flavivirus transmis par les moustiques comme le virus du Nil occidental, le virus de la dengue, le virus de la fièvre jaune, le virus de l’encéphalite japonaise et le virus Zika constituent des préoccupations croissantes de santé publique. Ils se sont répandus dans le monde au cours des dernières décennies, et les épidémies sont devenues plus fréquentes et plus sévères. Chaque année, des millions de personnes sont infectées et environ 50 000 patients décèdent d’infections à Flavivirus. Malgré les nombreux efforts de recherche, il n’y a actuellement aucun médicament antiviral spécifique disponible, et des nouvelles stratégies antivirales sont indispensables. Comprendre comment les Flavivirus fonctionnent au niveau moléculaire aidera à découvrir des nouvelles cibles pour l'intervention thérapeutique. Les Flavivirus ont un génome d'ARN simple brin de polarité positive qui code pour trois protéines structurales et huit protéines non structurales. Seules deux des huit protéines non structurales ont des activités enzymatiques. NS3 possède un domaine protéase et un domaine hélicase, et NS5 a un domaine méthyl- et guanylyltransférase et un domaine ARN polymérase ARN-dépendante. Ensemble, ils répliquent le génome viral. Ici, nous caractérisons l'interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive. Un modèle d'interaction comprenant NS3, NS5 et l’ARN viral a été développé basé sur des structures cristallines connues ainsi que des activités enzymatiques des deux protéines individuelles, et ce modèle a été soumis à des simulations de dynamique moléculaire. Les interactions potentielles entre les protéines NS3 et NS5 ont été identifiées. Les résidus impliqués dans ces interactions ont été mutés dans un réplicon du virus du Nil occidental et les effets de ces mutations sur la réplication virale ont été évalués. Une région particulière à la surface de la protéine NS3 a été identifiée comme étant cruciale pour la réplication virale, très probablement parce qu'elle interagit avec NS5. Cette région pourrait être une cible attrayante pour la recherche de composés qui pourraient interférer avec l'interaction entre NS3 et NS5 et donc posséder un potentiel antiviral intéressant. / Abstract : Mosquito-borne Flaviviruses like West Nile virus, Dengue virus, Yellow Fever virus, Japanese encephalitis virus, and Zika virus are increasing public health concerns. They have spread globally during the past decades, and outbreaks have recently become more frequent and more severe. Every year, millions of people are infected, and approximately 50,000 patients die from Flavivirus infections. Despite extensive research efforts, there are currently no specific antiviral drugs available, and new antiviral strategies are greatly needed. Understanding how Flaviviruses work on a molecular level will help in uncovering new points for therapeutic intervention. Flaviviruses have a single-stranded RNA genome of positive polarity that encodes three structural and eight non-structural proteins. Only two of the eight non-structural proteins have enzymatic activities. NS3 has an N-terminal protease domain and a C-terminal helicase domain, and NS5 has an N-terminal capping enzyme domain and a C-terminal RNA-dependent RNA polymerase domain. Together, they replicate the viral genome. Here we characterize the NS3:NS5 interaction within the West Nile virus RNA replicase complex during positive strand synthesis. An interaction model including NS3, NS5 and viral RNA was developed based on the known crystal structures as well as enzymatic activities of the two individual proteins, and this model was subjected to molecular dynamics simulations. Potential interactions between the NS3 and NS5 proteins were identified. Residues involved in these interactions were mutated in a West Nile virus replicon, and the effects of these mutations on viral replication were evaluated. One particular region on the surface of the NS3 protein was identified to be crucial for viral replication, most likely because it mediates the interaction with NS5. This region might be an attractive target for the search of compounds that could interfere with the NS3:NS5 interaction and therefore possess an interesting antiviral potential.

Le virus de la dengue cause une maladie de type grippal qui peut dans certains cas évoluer vers des fièvreshémorragiques mortelles. Mon projet de thèse porte sur la réplication de ce virus. Je focalise sur la compréhension du mécanisme d'action de la protéine NS5 de ce virus. La protéine contient 2 domaines : 1) domaine méthyltransférase, essentiel pour la traduction des protéines virales, 2) domaine polymérase, synthétisant le génome ARN du virus. Premièrement, nous avons démontré que la polymérase joue un rôle principal dans la conservation de l'extrémité 3' et 5' du génome et de l'anti-génome. Puis, j'ai caractérisé l'influence du domaine méthyltransférase sur l'activité polymérase de la protéine NS5. J'ai développé un système d'études mécanistiques en utilisant des techniques biochimiques de cinétique pré-stationnaire pour la protéine NS5, et obtenu des paramètres cinétiques et thermodynamiques de cette protéine envers ses substrats. Avec ce même système, j'ai pu tester des activités de la polymérase NS5 avec des ARN coiffés et triphosphates de différente longueur, mimant les séquences à l'extrémité 5' du génome du virus de la dengue. L'activité polymérase de NS5 est influencée par la présence de la coiffe de l'ARN, ce qui m'a permis de proposer une distance physique correspondant à environ 13 nucléotides entre les sites actifs domaines méthyltransférase et polymérase. Mes travaux ouvrent la voie à la détermination de la structure 3D de NS5 avec ses ARN et des nucléotides 5'-triphosphate.Elucider son mécanisme d'action, c'est être capable d'inhiber son action et donc de pouvoir proposer des molécules capables d'arrêter la prolifération virale lors d'une infection. / Dengue virus causes dengue fever, which may evolve towards life-threatening hemorrhagic fever. My research projectfocuses on dengue replication, and more precisely on the mechanism of NS5 at the molecular/atomic level. NS5 is a bifunctionalenzyme containing two domains: 1) a methyltransferase domain essential for translation of viral proteins, 2) apolymerase domain synthesizing the viral RNA genome. First, we demonstrated the main role of the polymerase in theconservation of 5' and 3' ends of dengue genome and anti-genome RNAs. Next, I showed the influence of themethyltransferase domain on the activity of the polymerase domain. I also developed a system allowing mechanistic studiesusing pre-steady state kinetics to characterize NS5 in depth. I have made use of this system to determine the catalyticparameters of NS5 towards its substrates. Using the same pre-steady state system, I was able to test the polymerase activityof NS5 with capped and uncapped 5'-triphosphate RNAs of different lengths corresponding to the 5'-end of the dengue RNAgenome. The polymerase activity of NS5 is significantly affected by the presence of the 5'-cap, which allowed me to designan experimental set-up pointing to a minimal physical distance of around 13 nucleotides between the methyltransferase andpolymerase active sites. My work will be useful to characterize the biophysics of NS5 in complex with its RNA and NTPsubstrates, and then to determine the crystal structure of such complex at play during viral RNA synthesis. Knowing thedetailed NS5 mechanism paves the way to inhibit its action and thus design drugs aiming at stopping a viral infection.

Au cours de ma thèse je me suis intéressée aux interactions protéine-protéine (PPI’s). Les PPI’s jouent un rôle majeur dans une grande diversité de processus cellulaires et sont maintenant considérées comme une cible majeure dans le but de développer de nouveaux médicaments. Cependant, cibler ce type d’interactions requiert le développement de chimiothèques dédiées, permettant d’accélérer la découverte de molécules « touches ». Pour surmonter ce problème, une chimiothèque orientée PPI (2P2I3D) a été conçu au laboratoire. Dans un premier temps, j’ai donc évalué cette chimiothèque sur différents complexes possédant des interfaces variées. Les résultats obtenus ont révélé des taux de touches supérieurs à ceux obtenus avec des chimiothèques non orientées, de 0,2 à 1,6% contre 0,01 à 0,1%, respectivement. Cette étude a permis d’établir une preuve de concept de la faisabilité de créer une chimiothèque orientée PPI, permettant ainsi une accélération de la découverte de composés biologiquement actifs.Dans un deuxième temps, je me suis intéressée à l’interaction entre deux protéines majeures du virus de la dengue : les protéines NS3 et NS5. J’ai tout d’abord identifié et caractérisé un nouveau site d’interaction, ce qui m’a permis de mettre en évidence que cette interaction avait pour conséquence d’augmenter l’activité enzymatique du domaine hélicase. J’ai par la suite recherché et identifié des petites molécules chimiques capable d’inhiber cette interaction. Les différentes caractérisations effectuées ont permis de mettre en évidence un effet antiviral. Ces inhibiteurs constituent un excellent point de départ afin d’étudier plus en détail le rôle biologique de ce complexe. / In my thesis I became interested in protein-protein interactions (PPI's). PPI's play a major role in a variety of cellular processes and are now considered a major target in order to develop new drugs. However, targeting such interactions requires the development of dedicated libraries, to accelerate the discovery of “hits”molecules .To overcome this issue, a focused chemical library PPI (2P2I3D) was designed in the laboratory.At first, I evaluated this chemical library on different complexes with diverse interfaces. The results showed higher hit rate to those obtained with non-oriented libraries, from 0.2 to 1.6% against 0.01 to 0.1%, respectively. This study has established a proof of concept of the feasibility of creating a focused chemical library PPI, thus accelerating the discovery of biologically active compounds.Secondly, I am interested in the interaction between two major proteins of dengue virus: the NS3 and NS5 proteins. I initially identified and characterized a novel interaction site, which allowed me to demonstrate that this interaction had the effect of increasing the enzymatic activity of the helicase domain. I searched and identified small molecules able to inhibit this interaction. The different characterizations helped to highlight an antiviral effect. These inhibitors are an excellent starting point to further explore the biological role of this complex.

Made available in DSpace on 2016-01-26T12:51:42Z (GMT). No. of bitstreams: 1 arielifernandagavioligomes_dissert.pdf: 1557143 bytes, checksum: 898e0714fcf73f6bc8ba53f5719efc71 (MD5) Previous issue date: 2011-12-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction: The Yellow Fever is characterized by severe hepatitis, renal failure, hemorrhage, and rapid terminal events that lead to shock and death. This disease is caused by the infection with the Yellow Fever Virus (YFV), considered the prototype of the Flavivirus genus. Its mechanism of replication is not well known but includes interactions of viral RNA with cellular and viral proteins. The nonstructural protein 5 (NS5) is the largest and most conserved protein of the Flavivirus genus; it encodes RNA-dependent RNA polymerase (RdRp) domains, besides possessing many important functions during viral replication, such as genic regulation of host cells. The protein hSlu7 is a homologous human protein, which was isolated interacting with the U5 that is involved in the second the step of alternative splicing. The hSlu7 is a predominantly nuclear protein and participates at the alternative splicing, influencing the correct choice of the alternative AGs of exon 3' so that the spliceossome is able to bind and start alternative splicing. Objective: To characterize the interaction of the hSlu7 protein with the YFV-NS5 protein and its cellular localization during viral infection. Material and Method: We confirmed the interaction of various NS5 with human proteins by two-hybrid assays. Deletion mutants were constructed and co-transformed with hSlu7 in yeast to determine the minimal domain of NS5 required for interaction. The cellular localization of the hSlu7 fused with GFP during the response of vaccine strain 17D of YFV in cells Vero E6, marked with anti-NS4AB and anti-NS5 for detection of the infection was also tested. Results: hSlu7 interacts with initial and final portions of the RdRp and the cytoplasmic sublocalization of hSlu7 occurs in the cells infected with YFV. Conclusions: Our results suggest that hSlu7 interacts with the YFV by two-hybrid system and the cellular sublocalization occurs due to the presence of viral infection. Further studies using RNA interference should be addressed to confirm the cellular function of hSlu7 , and to evaluate which alterations that infected and uninfected cells will suffer with low levels of hSlu7. / Introdução: A Febre Amarela é uma doença decorrente da infecção pelo Vírus da Febre Amarela (YFV) que é um protótipo do gênero Flavivirus que provoca uma severa hepatite, falência renal, hemorragia, e eventos que rapidamente levam ao choque e morte do indivíduo. Os mecanismos de replicação genômico do YFV não são bem conhecidos. A proteína não estrutural 5 (NS5) é a maior proteína e a mais conservada dos Flavivirus, ela codifica a RNA polimerase dependente de RNA (RdRp). A hSlu7 é uma proteína celular, predominantemente nuclear, e foi isolada interagindo com a U5 no segundo passo do splicing alternativo. A hSlu7 auxilia na correta seleção dos AGs alternativos do exon 3 para a realização da reação de splicing. Objetivo: Caracterizar a interação de hSlu7 com a proteína NS5 de YFV, quanto a sua localização celular durante a infecção. Material e Método: Pelo sistema duplo-híbrido em leveduras utilizando plasmid-linkage avaliamos a interação de hSlu7 com deleções mutantes da RdRp de YFV, e a localização celular de GFP-hSlu7 durante a replicação da cepa vacinal 17D de YFV em cultura de células Vero E6, marcadas com anticorpos NS4AB e NS5 para detecção da infecção. Resultados: A hSlu7 interage com as porções inicial e final da RdRp, e a sublocalização citoplasmática de hSlu7, ocorre nas células infectadas com YFV. Conclusão: Nossos resultados sugerem que a hSlu7 possui uma sublocalização celular durante a replicação do YFV, além de interagir com a RdRp viral. Ainda será necessária a confirmação da função celular de hSlu7 utilizando RNA de interferência para avaliar quais as alterações que a célula não infectada e infectada sofrerá diante dos níveis baixos de hSlu7.

Made available in DSpace on 2016-01-26T12:51:47Z (GMT). No. of bitstreams: 1 anacarolinabterzian_tese.pdf: 3328386 bytes, checksum: 0bae9bd7c5453a781cfbba32fac30196 (MD5) Previous issue date: 2013-11-08 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Introduction. Yellow Fever Virus (YFV) causes a hemorrhagic fever and it is the prototype of genus Flavivirus. Kunjin virus (KUNV) is naturally attenuated and is used to develop vaccine candidates against more pathogenic WNV strains. Flavivirus replication is a complex mechanism that involves interaction between viral RNA and cellular and viral proteins. The NS5 protein is the largest and highly conserved viral protein and it is critical for many functions, including replication, RNA capping and virus-host interactions. Once protein-protein interactions present basic importance for the activation, the regulation and the control of diverse enzymatic functions related to these interactions, the identification and the characterization of them are essential for a better comprehension of the pathogenesis and for the rational design of drugs for YFV. Previously, it was identified that the cellular protein p54nrb/NonO interacts with the RNA dependent RNA polymerase domain of YFV NS5. The p54nrb/NonO protein is a nuclear transcription factor associated with nuclear membrane and exhibits multifunction characteristics in nuclear processes in eukaryotic cells, in frequent association with the U1A and PSF proteins. Interaction between NS5 and p54nrb/NonO may influence localization and transport of proteins and viral RNA within the cell. Objective. The purpose of this study was to confirm the interaction between p54nrb/NonO and YFV and KUNV NS5 and determine the role of p54nrb/NonO on viral replication. Material and Method. Co-immunoprecipitation, mass spectrometry and indirect immunofluorescence assays were realized to confirm the interaction and co-localization between the proteins. To determine the effect on viral replication, the p54nrb/NonO and PSF were overexpressed in cellular culture, as well, the silencing of p54nrb/NonO. After, the replication level was determined by Tempo Real PCR, plaque assay, measuring of β-galactosidase and luciferase activity assays. Results. Immunofluorescence assays showed co-localization of p54nrb/NonO with YFV NS4 in the perinuclar region and with NS5 in the nucleus. In contrast, KUNV NS5 co-localized with p54nrb/NonO in the perinuclear region and co-precipitated with p54nrb/NonO. The co-precipitation between p54nrb/NonO and NS5 YFV was not identified. Again, it was identified by mass spectrometry analysis the co-precipitation of p54nrb/NonO by monoclonal antibodies to KUNV NS5 protein. The p54nrb/NonO overexpression did not affect the YFV and KUNV replication, however, PSF overexpression showed inhibitory effect on viral replication. The RNA interference assays were inconclusive about the role of p54nrb/NonO silencing on YFV replication. Conclusion. p54nrb/NonO and KUNV NS5 interact physically and co-localize in the cytoplasm, while, the co-localization with YFV NS5 occcurs in the nucleus, although, there is no physical interaction between them. However, the overexpression of p54nrb/NonO does not affect the viral replication. PSF was confirmed as an interactive partern of p54nrb/NonO and, when it is overexpressed, it inhibits YFV and KUNV replication. / Introdução. O vírus da Febre Amarela (YFV) causa febre hemorrágica e é o protótipo do gênero Flavivirus. O vírus Kunjin (KUNV) é naturalmente atenuado e usado para o desenvolvimento de candidatos vacinais contra linhagens mais patogênicas do WNV. A replicação do Flavivirus é um mecanismo complexo que envolve interações entre o RNA viral e proteínas virais e celulares. A NS5 é a maior e mais conservada proteína viral e é crítica para muitas funções, incluindo replicação, capeamento do RNA e interação vírus-hospedeiro. Como interações proteicas são de fundamental importância para ativação, regulação e controle de diversas funções enzimáticas a elas relacionadas, fica clara a relevância da identificação e caracterização das interações participantes desse processo para uma melhor compreensão da patogênese e para o desenho racional de drogas para a febre amarela. Foi identificado previamente que a proteína celular p54nrb/NonO interage com o domínio RNA polimerase RNA dependente de NS5 de YFV. p54nrb/NonO é um fator de transcrição nuclear associada a membrana nuclear e apresenta características multifuncionais nos processos celulares em células eucariotas, ocorrendo frequentemente em associação com as proteínas U1A e PSF. Dessa forma, a interação entre p54nrb/NonO e NS5 pode influenciar a localização, transporte das proteínas e do RNA viral dentro da célula. Objetivo. O objetivo deste estudo foi confirmar a interação entre p54nrb/NonO e NS5 de YFV e KUNV, e determinar o efeito da interação sobre a replicação viral. Materiais e Métodos. Para tanto, experimentos de co-imunoprecipitação, espectrometria de massa e imunofluorescência indireta foram realizados para confirmar a interação e a co-localização entre as proteínas. Para determinar o efeito sobre a replicação viral, foi realizado, em cultura celular, a superexpressão p54nrb/NonO e PSF, bem como, o silenciamento de p54nrb/NonO. Posteriormente, a taxa de replicação viral foi determinada por técnicas de qPCR, ensaio de placa, mensuração da atividade de β-galactosidase e luciferase. Resultados. O ensaio de imunofluorescência mostrou co-localização entre p54nrb/NonO e NS4 de YFV na região perinuclear e com NS5 no núcleo. Em contraste, NS5 de KUN co-localizou com p54nrb/NonO na região perinuclear, e da mesma forma, NS5 de KUNV foi identificado co-precipitando p54nrb/NonO. Não foi identificada a co-precipitação entre p54nrb/NonO e NS5 de YFV. Novamente, p54nrb/NonO foi identificada co-precipitando com NS5 de KUNV pela análise por espectrometria de massa com o uso de anticorpo monoclonal para a proteína NS5 de KUNV. A superexpressão de p54nrb/NonO não mostrou afetar a replicação de YFV e KUNV, entretanto, a superexpressão de PSF mostrou efeito inibitório sobre a replicação viral. Os estudos com interferência de RNA, contudo, foram inconclusivos sobre o efeito do silenciamento de p54nrb/NonO sobre a replicação de YFV. Conclusão. p54nrb/NonO e NS5 de KUNV interagem fisicamente e co-localizam no citoplasma, enquanto que, a co-localização com NS5 de YFV ocorre no núcleo, embora não ocorra interação física. Entretanto, a superexpressão de p54nrb/NonO não afeta a replicação viral. PSF foi confirmada como parceira interativa de p54nrb/NonO e, quando superexpressa, inibe a replicação de YFV e KUNV.

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