O vírus de Leishmania 1-4 ( do inglês Leishmania RNA virus 1-4 ou LRV1-4) é um vírus da família Totiviridae, e que possui capsídeo icosaédrico e RNA dupla-fita que codifica duas proteínas (proteína capsidial e RNA polimerase). Dados recentes indicam o envolvimento do LRV1-4 na patogênese de Leishmania no hospedeiro humano, tornando seu estudo de fundamental importância para o entendimento dessa doença e de seu papel na relação parasito-hospedeiro. Há relatos sobre a purificação do vírus a partir do seu hospedeiro natural (Leishmania guyanensis) e a partir de sistemas de expressão heteróloga. Este trabalho tem por objetivo estabelecer os métodos de purificação para posteriores estudos estruturais por Microscopia Eletrônica de Transmissão por Contraste Negativo (NS-TEM) e por Crio-Microscopia Eletrônica (Cryo-EM). Os estudos aqui propostos irão permitir a construção de um modelo estrutural do capsídeo do LRV1-4 e sua identificação correta dentre os totivírus. Além das contribuições ao conhecimento da biologia/patogenia do LRV1-4 este estudo representa a primeira caracterização estrutural de um capsídeo viral realizada no Brasil, e assim um avanço importante para a área de virologia e biologia estrutural no pais. Foram realizadas ultracentrifugações biológicas, utilizando gradientes de sacarose, para a purificação do vírus a partir do extrato celular de L. guyanensis. As frações que apresentaram RNA viral foram analisadas por Microscopia Eletrônica de Transmissão (Campinas LNNano CNPEM). Além disso, foram realizadas tentativas de expressar a proteína do capsídeo (ORF2) em Leishmania tarentolae e Escherichia coli. Foram também realizados esforços para a obtenção de anticorpos a partir de peptídeos sintetizados após análise computacional da sequência de aminoácidos da proteína do capsídeo. As amostras obtidas a partir do hospedeiro natural do vírus se apresentaram heterogêneas quando analisadas por NS-TEM, de modo que não foi possível a realização de uma análise estrutural. Porém, a presença de partículas do tamanho esperado para o vírus em amostras em que foi detectado o RNA viral indicam que são necessários esforços para obtenção de uma amostra de maior pureza e homogênea. Além disso, não foi possível obter a proteína do capsídeo nos sistemas de expressão heteróloga. A presença de 25 resíduos de cisteína pode estar levando a proteína à degradação rápida em bactéria. Os experimentos de expressão em células de Leishmania ainda não foram conclusivos. Foi obtido um anticorpo anti-peptídeo que reconhece a proteína do capsídeo, tornando possíveis experimentos como imunolocalização e imunoprecipitação do vírus. / The Leishmania RNA virus 1-4 (LRV1-4) belongs to the Totiviridae family. It has an icosahedral capsid and a double-strand RNA encoding two proteins (capsid protein and RNA polymerase). Recent data indicate the involvement of LRV1-4 in the pathogenesis of Leishmania in the human host, making their study of fundamental importance for the understanding of this disease and its role in host-parasite relationship. There are reports on the purification of the virus from its natural host (Leishmania guyanensis) and from the same heterologous expression systems such as Escherichia coli.This work aims to stablish purification methods for further structural studies by Negative Stain Transmission Microscopy (NS-TEM) and Cryo-Electron Microscopy (Cryo-EM). The studies proposed here will allow the construction of a structural model of the coat protein of LRV1-4 and their correct identification amongst the Totiviridae. In addition to the contributions to the knowledge of the biology and pathogenesis of LRV1-4, this study represents the first structural characterization of a viral capsid held in Brazil and thus an important step forward for the field of virology and structural biology in the country. Sucrose for virus purification gradients were performed from the cell extract of L. guyanensis. Fractions that showed viral RNA were analised by Transmission Electron Microscopy (Campinas LNNano - CNPEM). Furthermore, attempts have been made to express the capsid protein (ORF2) in Leishmania tarentolae and Escherichia coli. There has also been made efforts to obtain antibodies from peptides synthesized accordingly to the computer analysis of the amino acid sequence of the capsid protein. The samples obtained from the natural host of the virus showed a heterogeneous distribution of particles when examined by NS-TEM so that it was not possible to perform a structural analysis. However, the presence of particles of the size expected for the virus particles in samples where the viral RNA was detected indicate that efforts are necessary to obtain a more homogeneous and pure sample. Moreover, it was not possible to obtain the capsid protein in heterologous expression systems. The presence of 25 cysteine residues could have led to the protein rapid degradation in the bacteria host. The expression experiments in Leishmania cells were not yet conclusive. It was also possible to obtain an anti-peptide antibody recognizing the capsid protein, enabling immunoprecipitation and immunolocalization experiments.

O vírus de Leishmania 1-4 ( do inglês Leishmania RNA virus 1-4 ou LRV1-4) é um vírus da família Totiviridae, e que possui capsídeo icosaédrico e RNA dupla-fita que codifica duas proteínas (proteína capsidial e RNA polimerase). Dados recentes indicam o envolvimento do LRV1-4 na patogênese de Leishmania no hospedeiro humano, tornando seu estudo de fundamental importância para o entendimento dessa doença e de seu papel na relação parasito-hospedeiro. Há relatos sobre a purificação do vírus a partir do seu hospedeiro natural (Leishmania guyanensis) e a partir de sistemas de expressão heteróloga. Este trabalho tem por objetivo estabelecer os métodos de purificação para posteriores estudos estruturais por Microscopia Eletrônica de Transmissão por Contraste Negativo (NS-TEM) e por Crio-Microscopia Eletrônica (Cryo-EM). Os estudos aqui propostos irão permitir a construção de um modelo estrutural do capsídeo do LRV1-4 e sua identificação correta dentre os totivírus. Além das contribuições ao conhecimento da biologia/patogenia do LRV1-4 este estudo representa a primeira caracterização estrutural de um capsídeo viral realizada no Brasil, e assim um avanço importante para a área de virologia e biologia estrutural no pais. Foram realizadas ultracentrifugações biológicas, utilizando gradientes de sacarose, para a purificação do vírus a partir do extrato celular de L. guyanensis. As frações que apresentaram RNA viral foram analisadas por Microscopia Eletrônica de Transmissão (Campinas LNNano CNPEM). Além disso, foram realizadas tentativas de expressar a proteína do capsídeo (ORF2) em Leishmania tarentolae e Escherichia coli. Foram também realizados esforços para a obtenção de anticorpos a partir de peptídeos sintetizados após análise computacional da sequência de aminoácidos da proteína do capsídeo. As amostras obtidas a partir do hospedeiro natural do vírus se apresentaram heterogêneas quando analisadas por NS-TEM, de modo que não foi possível a realização de uma análise estrutural. Porém, a presença de partículas do tamanho esperado para o vírus em amostras em que foi detectado o RNA viral indicam que são necessários esforços para obtenção de uma amostra de maior pureza e homogênea. Além disso, não foi possível obter a proteína do capsídeo nos sistemas de expressão heteróloga. A presença de 25 resíduos de cisteína pode estar levando a proteína à degradação rápida em bactéria. Os experimentos de expressão em células de Leishmania ainda não foram conclusivos. Foi obtido um anticorpo anti-peptídeo que reconhece a proteína do capsídeo, tornando possíveis experimentos como imunolocalização e imunoprecipitação do vírus. / The Leishmania RNA virus 1-4 (LRV1-4) belongs to the Totiviridae family. It has an icosahedral capsid and a double-strand RNA encoding two proteins (capsid protein and RNA polymerase). Recent data indicate the involvement of LRV1-4 in the pathogenesis of Leishmania in the human host, making their study of fundamental importance for the understanding of this disease and its role in host-parasite relationship. There are reports on the purification of the virus from its natural host (Leishmania guyanensis) and from the same heterologous expression systems such as Escherichia coli.This work aims to stablish purification methods for further structural studies by Negative Stain Transmission Microscopy (NS-TEM) and Cryo-Electron Microscopy (Cryo-EM). The studies proposed here will allow the construction of a structural model of the coat protein of LRV1-4 and their correct identification amongst the Totiviridae. In addition to the contributions to the knowledge of the biology and pathogenesis of LRV1-4, this study represents the first structural characterization of a viral capsid held in Brazil and thus an important step forward for the field of virology and structural biology in the country. Sucrose for virus purification gradients were performed from the cell extract of L. guyanensis. Fractions that showed viral RNA were analised by Transmission Electron Microscopy (Campinas LNNano - CNPEM). Furthermore, attempts have been made to express the capsid protein (ORF2) in Leishmania tarentolae and Escherichia coli. There has also been made efforts to obtain antibodies from peptides synthesized accordingly to the computer analysis of the amino acid sequence of the capsid protein. The samples obtained from the natural host of the virus showed a heterogeneous distribution of particles when examined by NS-TEM so that it was not possible to perform a structural analysis. However, the presence of particles of the size expected for the virus particles in samples where the viral RNA was detected indicate that efforts are necessary to obtain a more homogeneous and pure sample. Moreover, it was not possible to obtain the capsid protein in heterologous expression systems. The presence of 25 cysteine residues could have led to the protein rapid degradation in the bacteria host. The expression experiments in Leishmania cells were not yet conclusive. It was also possible to obtain an anti-peptide antibody recognizing the capsid protein, enabling immunoprecipitation and immunolocalization experiments.

The membrane proteins signal peptide peptidase, signal peptide peptidase like and presenilin are intramembrane aspartyl proteases located in the endoplasmic reticulum, plasma membrane and organelle. These membrane proteins are able to catalyze a hydrolytic reaction in a hydrophobic space. The downstream consequences of these reactions impact a variety of cellular functions such as cytokine production, inflammatory responses, embryogenesis, and immune system regulation. Additionally, the aspartyl proteases such as signal peptide peptidase and presenilin, a part of the γ-secretase complex, hydrolyze peptides leading to pathogen maturation and Alzheimer’s disease, respectively. Electron crystallography offers the unique aspect of studying membrane proteins in a near native state. Determining the structures of Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases by electron crystallography may provide insight into how a hydrolysis reaction occurs in a hydrophobic environment and how the protein determines which transmembrane signal peptides to cleave. Additionally, structure determination may help answer questions regarding why human presenilin, part of the γ-secretase complex, incorrectly processes amyloid precursor protein into amyloid-beta peptides leading to Alzheimer’s disease. Such structural data may not only shed light on how amyloid precursor protein is processed but how other proteins are processed by signal peptide peptidase leading to immune responses, cell signaling, and pathogen maturation. In addition, structure-function data may have an impact on pharmaceutical drug designs that targets signal peptide peptidase, signal peptide peptidase like, and/or presenilin. To determine the structure of aspartyl proteases, two archaeal signal peptide peptidases were used for two-dimensional crystallization trials to be able to study their structure by electron crystallography. Haloarcula morismortui and Methanoculleus marisnigri JR1 signal peptide peptidases, both human signal peptide peptidase homologues, were recombinantly over-expressed and purified. During dialysis trials, various lipid-to-protein ratios, sodium chloride concentrations, temperatures, detergents and a variety of other variables were tested. Methanoculleus marisnigri JR1 signal peptide peptidase showed the most promising results in terms of crystallinity. Optimizing dialysis conditions, specifically narrowing the lipid to protein ratio, resulted in two-dimensional crystals. Ordered arrays measuring up to 200 nm x 200 nm were observed. These ordered arrays have been shown to be reproducible amongst multiple batches of purified Methanoculleus marisnigri JR1 signal peptide peptidase. Preliminary projection maps of negatively stained ordered arrays show unit cell dimensions of a = 178 Å, b = 160 Å, γ = 92.0 Å and a = 175 Å, b = 167 Å, γ = 92.0 Å. The monomer measurements are approximately 70 Å by 80 Å. This is the first time a signal peptide peptidase homologue has been crystallized by two-dimensional crystallization.

Le virus de la grippe est un virus à ARN négatif appartenant à la famille des Orthomyxoviridae qui se compose de 7 membres dont les virus influenza A, B, C et D. Le génome viral comprend 7 à 8 particules ribonucléoprotéiques (RNP) au sein desquelles l’ARN viral (ARNv) est recouvert de multiples copies de nucléoprotéines (NP) et est associé à l’ARN polymérase virale via ses extrémités 3’ et 5’. Au cours de ce travail de thèse, nous nous sommes tout d’abord focalisés sur l’étude biochimique de NP A et NP B et avons pu mettre en évidence des comportements différents en ce qui concerne leurs propriétés d’oligomérisation en présence ou en absence d’ARN et en fonction de la concentration en sel. Pour la première fois nous avons pu observer une structure similaire aux RNP mais reconstituée uniquement à partir de NP A et d’un ARN de 12 nucléotides. Nous avons pu formuler l’hypothèse que 12 nucléotides de l’ARN serait fixés à la NP avec une forte affinité tandis que le reste de l’ARN fixerait la NP avec une affinité beaucoup plus faible. En parallèle nous avons résolu la structure cristallographique de la nucléoprotéine de la grippe de type D et réaliser la caractérisation de son interaction avec l’importine-α7 humaine. Enfin nous avons étudié la fixation de l’ARN sur NP D et mis en évidence l’importance de l’extrémité C-terminale dans le processus de fixation à l’ARN. Ces informations ont permis de formuler de nouvelles hypothèses quant au fonctionnement du virus de la grippe et permettre d’inscrire ce projet de thèse dans une dynamique globale de lutte contre ce virus. / Influenza virus is a negative RNA virus belongs to the Orthomyxoviridae family which consists of 7 members including influenza viruses A, B, C and D. The viral genome comprises 7 to 8 ribonucleoprotein particles (RNP) in which the viral RNA (vRNA) is coated with multiple copies of nucleoproteins (NP) and is associated with the viral RNA polymerase by its 3 'and 5' ends. In this thesis, we first focused on the biochemical study of NP A and NP B and we demonstrate that there are different behaviors with regard to their oligomerization properties in the presence or absence of RNA and as a function of the salt concentration. For the first time we were able to observe a structure very similar to RNP which was reconstituted only from NP A and a 12 nucleotide RNA. Thus, we formulate the hypothesis that 12 nucleotides of the RNA would bind NP with a very strong affinity while the rest of the RNA would bind NP with a lower affinity. In parallel, we solved the crystallographic structure of the nucleoprotein of influenza D virus and we characterized its interaction with human importin-α7. Finally, we studied the binding of RNA on NP D and we demonstrated the importance of the C-terminal end in the RNA binding process. This thesis project made it possible to formulate new hypotheses concerning the functioning of the influenza virus and to include this thesis project in a global dynamic of combating the influenza virus.

<p>Human noroviruses (HuNoVs) are the leading cause of an acute form of non-bacterial gastroenteritis, where strains belonging to genogroup (G) II are dominant. Upon expression with the baculovirus culture system, virus-like particles (VLPs) of HuNoVs are expected to assemble into T = 3 icosahedral capsid particles resembling the structure of the infectious virion particles. However, some strains were found to assemble into either T = 1 or T = 4 capsids, or a combination of two different capsid forms. In this study, we showed that VLPs of the Virginia 1997 387 (VA387) GII.4 outbreak strain assembled into T = 1, T = 3, and T = 4 capsids upon expression in insect cell culture, the first case for a naturally occurring HuNoV strain to assemble into all three capsid states. TEM analysis revealed that T = 1 icosahedral particles were the most abundant in purified samples, which contrasts previous findings where either T = 3 or T = 4 were the most abundant. We resolved the cryo-EM structures of the T = 1 shell (S) domain, T = 3, and T = 4 particles to 2.24, 2.44, and 3.43 Å, respectively, making them the most resolved norovirus (NoV) structures to date. Single particle cryo-EM 3D analysis showed that the protruding (P) domain of T = 1 and T = 4 VLPs are highly dynamic. Additionally, we showed that T = 3 VLPs are resistant while T = 1 and T = 4 VLPs are sensitive to digestion in the presence of trypsin. This suggested that T = 1 and T = 4 capsids are less stable among the VLPs, which is consistent with the highly dynamic P domain inferred from our cryo-EM 3D analysis. During infection, HuNoVs travel through the gastrointestinal (GI) tract where they encounter a broad range of variable conditions that include pH, ionic strength, and host defenses (e.g., proteases). Our analyses suggest that virions are T = 3 particles as they can survive the GI tract upon exiting the host. We determined the first cryo-EM structure of T = 3 VLPs in complex with the known HuNoV host cell receptor, histo-blood group antigen (HBGA), to a resolution of 2.51 Å, demonstrating that NoV VLPs can serve as a platform in the structural characterization of small ligand molecules. Lastly, we identified a histidine residue retained in the S domain of all identified caliciviruses critical in the assembly of capsids. Our structures and their characterization will contribute to the development of therapeutic agents to combat noroviruses. </p>