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Repositório Da Universidade De Lisboa UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA DEPARTAMENTO DE MICROBIOLOGIA The structure-function relationship of HIV-1 Vif protein and its regulation Carla Iris Cadima Couto Tese orientada pelo Prof. Doutor João Gonçalves DOUTORAMENTO EM FARMÁCIA MICROBIOLOGIA 2008 As opiniões expressas neste trabalho são de exclusiva responsabilidade do seu autor. Carla Iris Cadima Couto was financially supported by a Ph.D scholarship SFRH/BD/18805/2004 from Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal. Preface The research work described in this dissertation was performed from October 2004 until September 2008 under the supervision of Prof. João Gonçalves at the CPM/URIA of the Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal. During the period of my research I spent three months (from September 2007 to December 2007) in Prof. Moshe Kotler laboratory at the Hadassah Medical School of the University of Jerusalém, Israel, and the work developed during this period is included in chapter 2 of this thesis and in a manuscript already submited. The results described in this thesis were included in manuscripts already submited for publication: Iris Cadima-Couto, Acilino Freitas-Vieira, Roni Nowarski, Elena Britan-Rosich, Moshe Kotler, and João Gonçalves. (2008). Modelling APOBEC3G intracellular steady-state shows a differential inhibition of HIV-1ΔVif. Journal of Biological Chemistry. (Submited) . Iris Cadima-Couto, Soraia Oliveira, Saraiva N, João Gonçalves. (2008). Cooperation of amino and carboxyl-terminal domains of Vif is essential for its role in A3G degradation. Journal of Biological Chemistry. (Submited) . Iris Cadima-Couto, Saraiva N, Gonçalves J. (2008). Assessment of HIV-1 Vif-APOBEC Interactions Based on a β-Lactamase Protein Fragment Complementation Assay. Retrovirology. (Submited) According to the “Decreto-Lei 388/70”, article 8 point nº 2, the data presented in this dissertation is the result of the authors work and it is clearly acknowledged in the text whenever data or reagents produced by others were used. The author participated in the planning and execution of the experimental procedures such as in the results interpretation and in manuscripts preparation. The opinions expressed in this publication are from the exclusive responsibility of the author and have not been previously submitted for any degree at this or other university. iii Acknowledgments/Agradecimentos Ao meu orientador, Professor João Gonçalves, pela sua orientação durante o desenvolvimento deste trabalho. Obrigada por todas as recomendações e por me ter dado liberdade na autogestão do meu tempo de trabalho no laboratório, que foi fundamental para que este trabalho tivesse sido concluído dentro dos prazos limites. Ao Professor José Moniz Pereira por permitir a realização desta tese na Faculdade de Farmácia da Universidade de Lisboa. À Fundação para a Ciência e Tecnologia pelo apoio financeiro no âmbito da bolsa de doutoramento, SFRH/BD/18805/2004. Ao Acilino por ter sido um excelente colega de trabalho, pelo seu companheirismo e amizade e por ter estado sempre presente durante os bons e maus momentos ao longo destes 4 anos. Obrigada por tudo! Aos meus restantes colegas de laboratório que me acompanharam ao longo deste trabalho: Ana Fonseca, Andreia, Cláudia, Filipa, Frederico, Maria João Leite, Maria João, Mariana, Paula, Rita, Sara, Sofia Coelho, Sofia CR, Soraia, Sylvie. A todos, obrigada! Obrigada a todos os meus restantes companheiros da unidade de microbiologia, com os quais partilhei as muitas horas no P3. À Madalena e ao João Vital pela sua simpatia e boa disposição. À Professora Aida Esteves, obrigada por ter disponibilizado o seu P3 durante um período do meu doutoramento. Obrigado também à Sandra, ao Rui, ao João e ao Ricardo, do grupo da Prof. Aida, por me terem sempre recebido tão bem. I am deeply grateful to Professor Moshe Kotler, from the University of Jerusalem, for receiving me so well in his lab, for his support and friendship and for making me feel at home away from home. I would also like to thank to all the members of Prof. Moshe lab: Roni and Elena, thank you so much for being so kind in your guidance during my work in the lab. Maia and Shira, thank for your friendship! iv À minha família, por me terem sempre apoiado em todas as decisões da minha vida. Obrigada aos meus amigos que acompanharam de perto todos os momentos do meu doutoramento….obrigada pela conversas, por todos os momentos de lazer que passámos e que foram essências durante este período. Finalmente, um especial agradecimento ao Rafael. Obrigada pelo teu constante apoio, paciência, e por nunca teres duvidado de que eu iria conseguir ultrapassar esta etapa da minha vida. Obrigada por me teres dado ânimo nos momentos mais difíceis deste doutoramento e por teres partilhado comigo todos os bons momentos. v Estrutura-Função da proteína Vif do HIV-1 e sua regulação SUMÁRIO O Vírus da Imunodeficiência Humana tipo 1 (HIV-1) é um retrovírus complexo que codifica uma proteína acessória, Vif (Viral Infectivity Factor), necessária para uma produtiva infecção. A presença de Vif na célula produtora de vírus é um dos factores essenciais para o aumento da infecciosidade viral, podendo esta ser aumentada até cerca de 1000 vezes. Há mais de uma década que é sabido que a proteína Vif é necessária para a replicação do HIV-1 em algumas células, chamadas não permissivas. No entanto em outras, chamadas permissivas, a replicação viral ocorre mesmo que na ausência de Vif. A razão para este facto permaneceu desconhecida até á descoberta, em 2002, da desaminase de citidina humana, human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) como sendo a proteína responsável por restringir a replicação de HIV-1 que não expressa Vif. Este factor de restrição celular encontra-se presente maioritariamente no baço, gónadas, monócitos e linfócitos periféricos. Na ausência de Vif, A3G, é encapsidado no virião em formação por um sistema dependente de Gag. Desta forma, A3G é transportado para a próxima célula alvo onde vai actuar no momento da transcrição reversa, desaminando a cadeia simples, negativa do cDNA viral. Este processo resulta na inactivação do processo replicativo por hipermutação do genoma ou mesmo pela degradação do cDNA viral por um mecanismo dependente da enzima DNA uracil glicosilase. Após a identificação de A3G e mais tarde de APOBEC3F (A3F), ficou claro que o mecanismo pelo qual o HIV-1 escapava á acção destes factores de restrição era através da proteína Vif. Vif actua inibindo a entrada de A3G e A3F nos viriões através da diminuição dos níveis celulares destes factores de restrição. Um dos mecanismos que a Vif utiliza implica a degradação proteossomal destas enzimas. Para isso, a Vif recruta uma série de proteínas celulares para formar um complexo Culina5- os desta forma para degradação proteossomal. vi Desta forma, torna-se essencial compreender de que forma a proteína A3G, documentada como sendo a que exerce maior actividade contra o HIV-1, pode ser estimulada na célula de maneira a poder escapar aos efeitos da Vif e consequentemente inibir a replicação viral. Assim, decidimos modelar o tempo de meia-vida do A3G como forma de monitorizar a sua acção na célula desde o momento em que é traduzido até á sua encapsidação no virião. Este mecanismo de modelação do tempo de meia-vida de uma proteína é conhecido pelo nome de “N-end Rule” e consiste na introdução de diferentes amino ácidos no inicio da região N-terminal de uma proteína resultando em diferentes tempos de meia-vida da mesma, de acordo com a identidade do resíduo introduzido. Ao interferir com a estabilidade desta proteína, fomos capazes de a direccionar para degradação no proteossoma duma forma independente da Vif. Os resultados apresentados neste trabalho confirmaram a importância do tempo de meia-vida do A3G para a manutenção da sua actividade antiviral. Ao diminuir os tempos de vida do A3G na célula, fomos capazes de determinar que o tempo de meia-vida mínimo do A3G para que este seja incorporado nos viriões é de aproximadamente 13 minutos. Neste caso, concluímos que a Vif terá que actuar nos estágios iniciais da vida do A3G de forma a impedir que este seja encapsidado nos viriões. A análise da actividade catalítica dos nossos diferentes A3Gs revelou que a sua actividade enzimática no citoplasma da célula não parece ser importante para a sua actividade antiviral e que a actividade catalítica destes variantes não aumenta de acordo com o aumento do tempo de meia-vida destes. Esta estratégia, permitiu-nos compreender de que forma o tempo de meia-vida do A3G pode influenciar a sua actividade antiviral, quer ao nível da sua encapsidação nos viriões como ao nível da sua actividade catalítica na célula. A identificação de padrões de interacção entre a Vif e o A3G é um dos maiores desafios para o desenvolvimento de novas terapias antivirais. Tem sido demonstrado que o impedimento da interacção Vif-A3G resulta num aumento da actividade antiviral deste último. Assim, decidimos estudar a interacção entre estas duas proteínas através de duas estratégias que, embora já tenham sido utilizadas em estudos semelhantes com outras proteínas, foram aqui usadas pela primeira vez no contexto da interacção Vif- A3G. vii A primeira estratégia consistiu na fusão de fragmentos da Vif e do A3G a domínios de interacção do factor de transcrição de levedura GCN4 (Zip), que forma homodimeros. A presença de Zip em fusão com estas proteínas obrigará estas a se encontrarem espacialmente permitindo-nos assim mutagenizar partes de ambas as proteínas e concluir acerca da importância de certos domínios na interacção. Esta estratégia permitiu-nos demonstrar a importância da região C-terminal da Vif na interacção com o A3G. Os resultados obtidos indicam que esta região da Vif, por si só, não é suficiente para induzir a degradação do A3G e sugerem que a interacção Vif-A3G deverá envolver um co-factor celular adicional que se deverá ligar á região N-terminal da Vif.
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