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Avaliação Dos Genes De Herpesvírus Bovino Tipo 5 No Controle Da Apoptose

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Avaliação Dos Genes De Herpesvírus Bovino Tipo 5 No Controle Da Apoptose UNIVERSIDADE DE BRASÍLIA FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE ANIMAL AVALIAÇÃO DOS GENES DE HERPESVÍRUS BOVINO TIPO 5 NO CONTROLE DA APOPTOSE ALICE MARTINS DA SILVA DISSERTAÇÃO DE MESTRADO EM SAÚDE ANIMAL BRASÍLIA-DF JULHO/2019 UNIVERSIDADE DE BRASÍLIA FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE ANIMAL AVALIAÇÃO DOS GENES DE HERPESVÍRUS BOVINO TIPO 5 NO CONTROLE DA APOPTOSE ALICE MARTINS DA SILVA ORIENTADOR: JOSÉ RENATO JUNQUEIRA BORGES DISSERTAÇÃO DE MESTRADO EM SAÚDE ANIMAL PUBLICAÇÃO 165/2019 BRASÍLIA/DF JULHO/2019 UNIVERSIDADE DE BRASÍLIA FACULDADE DE AGRONOMIA E MEDICINA VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE ANIMAL AVALIAÇÃO DOS GENES DE HERPESVÍRUS BOVINO TIPO 5 NO CONTROLE DA APOPTOSE Alice Martins da Silva DISSERTAÇÃO DE MESTRADO SUBMETIDA AO PROGRAMA DE SAÚDE ANIMAL COMO PARTE DOS REQUISITOS À OBTENÇÃO DO GRAU DE MESTRE EM SAÚDE ANIMAL. BRASÍLIA, DF, 10 DE JULHO DE 2019. REFERÊNCIA BIBLIOGRÁFICA E CATALOGAÇÃO Silva.A.M. Avaliação dos genes de herpesvírus bovino tipo 5 no controle da apoptose. Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília. UnB. 2019. 65p. Dissertação de mestrado. Documento formal autorizando reprodução desta dissertação de mestrado para empréstimo ou comercialização exclusivamente para fins acadêmicos. Foi passado pelo autor à Universidade de Brasília e acha-se arquivado na Secretaria do Programa. O autor reserva para si os outros direitos autorais de publicação. Nenhuma parte desta dissertação de mestrado pode ser reproduzida sem a autorização por escrito do autor. Citações são estimuladas desde que citada a fonte. FICHA CATALOGRÁFICA Silva, Alice Martins. Avaliação dos genes de herpesvírus bovino tipo 5 no controle da apoptose. Alice Martins da Silva; orientação de José Renato Junqueira Borges - Brasília, 2019. 65 p: il. Dissertação de Mestrado (M) – Universidade de Brasília/ Faculdade de Agronomia e Medicina Veterinária, 2019. 1. Alfaherpesvírus bovino tipo 5. 2. US3. 3. Apoptose I. Borges, J.R.J. II. Avaliação dos genes de herpesvírus bovino tipo 5 no controle da apoptose. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. iv DEDICATÓRIA À Ariane Abrunhosa e Luiz Martins da Silva. Meus companheiros de travessias. v AGRADECIMENTOS Ao Professor Dr. José Renato Junqueira por acreditar em mim e me proporcionar a liberdade de realizar o mestrado na área de Microbiologia. Agradeço às professoras Dra. Lígia Cantarino e Dra. Simone Perecmanis por terem me incentivado a realizar o mestrado e me ajudado a crescer desde a graduação. Ambas são para mim, uma referência de profissionalismo e dedicação. Ao Professor Dr. Bergmann Ribeiro pela paciência, cordialidade, ensinamentos e, principalmente, pelo exemplo de mestre e pesquisador. Agradeço pelas sábias palavras, quando tudo parecia não dar certo. Sem o seu apoio nada disso teria sido possível. Um agradecimento especial ao Professor Dr. Fabrício Souza Campos, pelo acompanhamento no desenvolvimento desse trabalho, pelas correções e pela paciência e confiança depositadas em mim. À Professora Dra. Ana Cláudia Franco, a Prof. Dra. Ângela Patrícia Santana e ao Prof. Dr. Renato Resende por aceitarem participar da banca. Agradeço a todos os técnicos e terceirizados do Hospital Veterinário de Pequenos Animais da UnB: Maurício Macêdo, Cléia Nunes, Ernane Paiva, Marcela Scalon, Bruno Dallago, Salvina, Célia, Dino, Léo e a toda equipe do Laboratório de Microbiologia Veterinária pela amizade, boa companhia e pelo café da tarde de todos esses anos. Aos doutores Leonardo Assis e Fabrício Morgado pessoas que me aconselharam, gastaram longas horas me explicando técnicas, refazendo experimentos, revisando e discutindo resultados. Ainda, agradeço a convivência e bons momentos com todos os colegas do Laboratório de Baculovírus e Laboratório de Virologia Vegetal: estagiários, mestrandos, pós- doutorandos, que de alguma maneira me ajudaram a amadurecer tanto profissionalmente como pessoalmente. Às amigas de uma vida, Luciana Santos, Sabrina Azevedo, Nanci Hokino, Letícia Muller que independente do momento ou da distância, sempre estiveram por perto. Agradeço ao meu namorado Juliano Terra que suportou a distância nesses anos e que esteve comigo em todos os momentos, me apoiando e encorajando a ser sempre melhor. A toda minha família, mas principalmente à minha incrível mãe, Ariane Abrunhosa, que me apoiou e nunca mediu esforços para que eu realizasse meus sonhos. Ao meu pai, Luiz Martins da Silva, meu primeiro professor, por ser tão amoroso e um exemplo de pessoa em tantos aspectos. Aos órgãos de fomento, FAPDF e CAPES que financiaram esse projeto. À Universidade de Brasília, essa incrível instituição, e a todos os seus funcionários e professores, muito obrigada! vi SUMÁRIO Página RESUMO VIII ABSTRACT IX LISTA DE ABREVIATURAS E SÍMBOLOS X 1. INTRODUÇÃO 1 2. OBJETIVOS 3 3. REVISÃO BIBLIOGRÁFICA 4 3.1. Alfaherpesvírus bovino tipo 5 4 3.2. Epidemiologia e controle 5 3.3. Etiologia – descrição do agente 6 3.4. Genoma 7 3.4.1. Ciclo Replicativo 8 3.4.2. Adsorção, fusão e penetração 9 3.4.3. Transporte e penetração nuclear 9 3.4.4. Expressão gênica e replicação do genoma 9 3.4.5. Morfogênese, maturação e egresso 10 3.4.6. Latência 11 3.4.7. Gene US3 13 3.5. Apoptose 15 3.6. Baculovírus: características gerais 17 3.6.1. Baculovírus no controle da morte celular programada 19 4. REFERÊNCIAS 22 5. INFORMAÇÕES ADICIONAIS 29 6. ARTIGO CIENTÍFICO 30 vii RESUMO AVALIAÇÃO DOS GENES DE HERPESVÍRUS BOVINO TIPO 5 NO CONTROLE DA APOPTOSE O Alphaherpesvirus bovino tipo 5 (BoHV-5) é um dos principais agentes responsáveis pela meningoencefalite em bovinos no Brasil, causando perdas econômicas significativas. Sabe-se que outros vírus da família Herpesviridae, como o Alphaherpesvirus bovino tipo 1, o Alphaherpesvirus suíno tipo 1 e o Alphaherpesvirus humano tipo 1 e 2, codificam genes homólogos ao BoHV-5, com reconhecida ação no controle da apoptose. O objetivo deste trabalho foi expressar o gene BoHV-5 US3 em um sistema de expressão baseado em baculovírus para a produção da proteína serina/treonina quinase e avaliar sua atividade no controle da apoptose in vitro. Um baculovírus recombinante derivado do Autographa californica multiple nucleopolyhedrovirus (AcMNPV) contendo o gene US3 e uma deleção no gene p35 anti-apoptótico de baculovírus foi construído usando o sistema Bac-to-BacTM. Este baculovírus recombinante foi utilizado para avaliar a atividade anti-apoptótica do gene US3 em células de inseto comparando-o com outros dois recombinantes de AcMNPV. Um vírus contendo uma cópia funcional do gene anti-apoptótico p35 de AcMNPV e outro AcMNPV p35 knockout com o gene iap-3 anti-apoptótico do vírus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). Descobrimos que o nível de caspase (indicador de apoptose) foi maior nas células de inseto infectadas com o vírus recombinante contendo o gene US3 do que nas células infectadas com os recombinantes AcMNPV com os genes p35 e iap-3. Além disso, o gene iap-3 AgMNPV foi capaz de substituir eficientemente o gene p35 no controle da apoptose durante a infecção de células de insetos pelo recombinante AcMNPV contendo o gene iap-3. Estes resultados indicam que o gene da proteína quinase BoHV-5 US3 não é capaz de bloquear a apoptose em células de inseto induzidas pela infecção de um AcMNPV p35 knockout. Palavras-chave: Alfaherpesvírus bovino tipo 5, US3, apoptose. viii ABSTRACT EVALUATION OF BOVINE TYPE 5 HERPESVIRUS GENES IN THE CONTROL OF APOPTOSIS Bovine alphaherpesvirus type 5 (BoHV-5) is one of the main agents responsible for meningoencephalitis in bovines in Brazil, causing significant economic losses. It is known that other viruses of the Herpesviridae family such as Bovine alphaherpesvirus type 1, Swine alphaherpesvirus type 1 and the Human alphaherpesvirus types 1 and 2 encode genes homologous to BoHV-5, with recognized action in the control of apoptosis. The objective of this work was to express the BoHV-5 US3 gene in a baculovirus based expression system for the production of the serine/threonine kinase protein and to evaluate its activity in the control of apoptosis in vitro. A recombinant baculovirus derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) containing the US3 gene and a deletion in the baculovirus anti-apoptotic gene p35 gene was constructed using the Bac-to-BacTM system. This recombinant baculovirus was used to evaluate the anti-apoptotic activity of the US3 gene in insect cells comparing with two other AcMNPV recombinants. One containing a functional copy of the AcMNPV anti-apoptotic p35 gene and an AcMNPV p35 knockout virus with the anti-apoptotic iap-3 gene from Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). We found that the caspase level (indicator of apoptosis) was higher in insect cells infected with the US3-contanining recombinant virus than in cells infected with the AcMNPV recombinants containing the p35 and iap-3 genes. Moreover, the AgMNPV iap-3 gene was able to efficiently substitute the p35 gene in the control of apoptosis during infection of insect cells by the AcMNPV recombinant containing the iap-3 gene. These results indicate that the BoHV-5 US3 protein kinase gene is not able to block apoptosis in insect cells induced by the infection of a p35 knockout AcMNPV. Key words: Alphaherpesvirus Bovine 5, US3, apoptosis. ix LISTA DE ABREVIATURAS E SÍMBOLOS AcMNPV:
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