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INTERAÇÕES TOSPOVÍRUS- PLANTA: Caracterização Estrutural De Proteínas De Tospovírus E Identificação De Interações Entre Proteínas Virais E Celulares

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INTERAÇÕES TOSPOVÍRUS- PLANTA: Caracterização Estrutural De Proteínas De Tospovírus E Identificação De Interações Entre Proteínas Virais E Celulares UNIVERSIDADE DE BRASÍLIA INSTITUTO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE BIOLOGIA CELULAR PÓS-GRADUAÇÃO EM BIOLOGIA MOLECULAR LABORATÓRIO DE VIROLOGIA VEGETAL INTERAÇÕES TOSPOVÍRUS- PLANTA: Caracterização estrutural de proteínas de tospovírus e identificação de interações entre proteínas virais e celulares Rayane Nunes Lima Tese de Doutorado apresentada ao Programa de Pós- Graduação em Biologia Molecular da Universidade de Brasília, para obtenção do título de Doutor em Biologia Molecular. Orientador: Dr. Renato de Oliveira Resende Brasília, 2018. 1 UNIVERSIDADE DE BRASÍLIA INSTITUTO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE BIOLOGIA CELULAR PÓS-GRADUAÇÃO EM BIOLOGIA MOLECULAR LABORATÓRIO DE VIROLOGIA VEGETAL INTERAÇÕES TOSPOVÍRUS-PLANTA: Caracterização estrutural de proteínas de tospovírus e identificação de interações entre proteínas virais e celulares Capítulo I: Introdução Geral Capítulo II: Homology modeling and molecular dynamics provide structural insights into tospovirus nucleoprotein Capítulo III: The complete genome of the tospovirus Zucchini lethal chlorosis virus and the structural analysis of its nucleocapsid protein (NP) Capítulo IV: Predição da estrutura terciária, Expressão e Purificação da proteína NSS de GRSV Capítulo V: Interações entre proteína virais e proteínas do hospedeiro 2 RAYANE NUNES LIMA INTERAÇÕES ORTHOTOSPOVÍRUS- PLANTA: Caracterização estrutural de proteínas de orthotospovírus e identificação de interações entre proteínas virais e celulares Banca Examinadora: _________________________________________ Prof. Dr. Renato de Oliveira Resende (Orientador) (CEL-UnB) _________________________________________ Prof. Dr. Tatsuya Nagata (CEL-UnB) _________________________________________ Dra. Marília Santos Silva (Embrapa-Recursos Genéticos e Biotecnologia) _________________________________________ Dra. Simone da Graça Ribeiro (Embrapa-Recursos Genéticos e Biotecnologia) _________________________________________ Prof. Dr. Bergmann Morais Ribeiro (Suplente) (CEL-UnB) 3 “We live in a society exquisitely dependent on Science and technology, in which, hardly anyone knows anything about Science and technology. This is a clear prescription for disaster.” “Vivemos em uma sociedade extremamente dependente da ciência e tecnologia, na qual pouquíssimos sabem alguma coisa sobre ciência e tecnologia. Isto é uma clara prescrição para o desastre.” Carl Edward Sagan, 1990.Why We Need To Understand Science. The Skeptical Inquirer, Vol. 14.3. 4 Índice Resumo .......................................................................................................................................... 8 Abstract ......................................................................................................................................... 9 Motivação.................................................................................................................................... 10 Justificativa .......................................................................................Erro! Indicador não definido. Objetivos ..................................................................................................................................... 11 Objetivo Geral ......................................................................................................................... 11 Objetivos Específicos ............................................................................................................... 11 CAPÍTULO I: Introdução Geral ..................................................................................................... 12 1.1 Ordem Bunyavirales .............................................................................................................. 13 1.1.1 Taxonomia e Diversidade ............................................................................................... 13 1.1.2 Vírions e Genoma ........................................................................................................... 16 1.2 Família Tospoviridae .............................................................................................................. 19 1.2.1 Taxonomia e diversidade ............................................................................................... 19 1.2.1 Vírions e Genoma ........................................................................................................... 21 1.2.3 Groundnut ringspot orthotospovirus (GRSV) ................................................................. 26 1.3 Ferramentas moleculares para o estudo das interações proteicas .................................. 27 1.3.1 Interações proteína-proteína para orthotospovírus ...................................................... 30 1.4 Ferramentas para predição in silico da estrutura tridimensional de proteínas.................... 33 1.5 Referências ............................................................................................................................ 34 CAPÍTULO II: Homology modeling and molecular dynamics provide structural insights into orthotospovirus nucleoprotein ................................................................................................... 42 Homology modeling and molecular dynamics provide structural insights into orthotospovirus nucleoprotein .............................................................................................................................. 43 2.1 Abstract ................................................................................................................................. 44 2.1.1 Background .................................................................................................................... 44 2.1.2 Results ............................................................................................................................ 44 2.1.3 Conclusions .................................................................................................................... 44 2.2 Background............................................................................................................................ 46 2.3 Results and Discussion .......................................................................................................... 47 2.3.1 Three-dimensional model of GRSV NP and Oligomerization ..................................... 47 2.3.2 RNA interaction .......................................................................................................... 52 2.4 Conclusions............................................................................................................................ 54 2.5 Methods ................................................................................................................................ 54 5 2.5.1 In silico Homology modeling and Model optimization .................................................. 54 2.5.2 Molecular dynamics ....................................................................................................... 55 2.6 Additional files:...................................................................................................................... 56 Additional file 1: The sequence codes of the viruses used at this work. ............................... 56 Additional file 2: Ramachandran plot analysis of predicted structure of Groundnut ringspot orthotospovirus (GRSV) NP protein. ........................................................................................ 56 2.7 Considerações. ...................................................................................................................... 57 2.8 References ............................................................................................................................. 58 CAPÍTULO III: The complete genome of the tospovirus Zucchini lethal chlorosis orthotospovirus and the structural analyses of its nucleocapsid protein (NP). .................................................... 63 3.1 Abstract ................................................................................................................................. 65 3.1.1 Background................................................................................................................. 65 3.1.2 Findings ...................................................................................................................... 65 3.1.3 Conclusion .................................................................................................................. 65 3.2 Body of text ........................................................................................................................... 66 3.2.1 Abbreviations ......................................................................Erro! Indicador não definido. 3.2.2 Competing interests ............................................................Erro! Indicador não definido. 3.2.3 Authors’ contribution ..........................................................Erro! Indicador não definido. 3.2.4 Acknowledgments ...............................................................Erro! Indicador não definido. 3.3 Análise da estrutura tridimensional da proteína do nucleocapsídeo (NP) do ZLCV ............. 73 3.3.1 Figura Suplementar: ....................................................................................................... 76 3.4 Referências ............................................................................................................................ 76 CAPÍTULO IV: Predição
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