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PB2 ist ein essentieller Bestandteil der trimeren RNA abhängigen RNA Polymerase von Influenzaviren und ist bekannt für seine Schlüsselrolle in der Bestimmung des Viruswirtsspektrums. Diese Arbeit diente der Identifizierung neuer Interaktionspartner von PB2 eines saisonalen und eines hochpathogenen Influenzavirus Stammes im Kontext infizierter humaner alveolar Epithelzellen (A549) unter Einsatz massenspektrometrischer Analysen. Die anschließende Untersuchung ausgewählter zellulärer Interaktoren hatte zum Ziel, deren Einfluss auf den Replikationszyklus der Influenzaviren zu bestimmen, sowie Unterschiede in ihrer Relevanz für das saisonale und das hochpathogene Virus aufzuzeigen. Die Erzeugung und Nutzung von Influenzaviren die einen Strep-tag an ihrem PB2 Protein tragen ermöglichte eine Anreicherung von PB2 und seiner Interaktionspartner. Die anschließende massenspektrometrische Analyse identifizierte 22 potentielle PB2 Interaktionspartner. Eine Auswahl an 13 Proteinen wurde tiefer gehend analysiert und eine Komplexbildung mit PB2 konnte für 9 Proteine bestätigt werden. Darüber hinaus zeigten 11 Proteine einen Polymerase stimulierenden bzw. hemmenden Effekt. Das Polymerase stimulierende Protein HSPA8 wurde zur weiteren Untersuchung ausgewählt. Während ein Einfluss von HSPA8 auf den hochpathogenen Influenzastamm nicht abschließend geklärt werden konnte, wurde seine Bedeutung für den Vermehrungszyklus des saisonalen Stammes aufgezeigt. Die Überexpression von HSPA8 führte zu einer Steigerung der Polymerase-Aktivität, wohingegen die Erniedrigung des HSPA8 Spiegels in einer Verringerung der viralen Replikation und der Polymerase-Aktivität resultierte. Interessanterweise führte die Erniedrigung des HSPA8 Spiegels auch zu stark verminderter PB2-Expression, jedoch nur im Falle des saisonalen Influenzastammes. Dieser Befund deutet auf eine Rolle von HSPA8 als PB2-Chaperon, notwendig für Proteinstabilität von saisonalen aber nicht hochpathogenen Influenzaviren, hin. / PB2 is an essential component of the influenza virus trimeric RNA dependent RNA polymerase and is known to play a key role in virus host range determination. Here, a combined affinity-purification/mass spectrometric approach was performed to identify novel interaction partners of PB2 of seasonal and highly pathogenic viral strains in infected human alveolar epithelial cells (A549). The subsequent analysis of selected cellular interaction partners aimed to determine the influence of these proteins on the replication cycle, as well as to determine differences in their relevance for the seasonal and the highly pathogenic influenza virus strain. Generation and use of recombinant influenza viruses carrying a Strep-tag at their PB2 protein allowed for enrichment of PB2 and its interaction partners. The subsequent mass spectrometric analysis identified 22 potential PB2 interaction partners. A selection of 13 proteins was further analyzed, and co-precipitation with PB2 was confirmed for 9 proteins. Moreover, an inhibitory or stimulatory effect on polymerase activity was observed for 11 proteins. The polymerase stimulating protein HSPA8 was selected for further investigation. While the influence of HSPA8 on the highly pathogenic strain remained unclear, its importance for seasonal influenza virus life cycle was demonstrated. Overexpression of HSPA8 resulted in increased polymerase activity while HSP8 knock down resulted in reduction of viral replication and viral polymerase activity. Intriguingly, the knock down of HSPA8 led to a strong decrease of PB2 protein expression. However, this was only observed for seasonal PB2. These results indicate a role of HSPA8 as a PB2 chaperone, necessary for protein stability of seasonal but not highly pathogenic influenza virus.

On estime que 10%-20% de la population mondiale est infectée chaque année par des virus influenza A (IAV) saisonniers, causant 250 à 500 000 morts. De plus ces virus présentent des risques de pandémie, et sont à ce titre un problème de santé publique majeur. Le cycle viral est dépendant de la capacité du virus à manipuler le protéome cellulaire. Par ailleurs, le système ubiquitine-protéasome (SUP) cellulaire est impliqué dans de nombreux processus de régulation cellulaires par l'induction de la dégradation de protéines, ou par la modification de leur activation ou de leur localisation sub-cellulaire. Le SUP est une cible privilégiée des virus lors de l'infection. Des études récentes indiquent qu'un réseau d'interactions entre les protéines virales des IAV et les protéines du SUP pourrait contribuer à la réplication virale et l’échappement du virus face au système immunitaire. Cependant ces interactions restent encore mal connues. Nous avons construit une banque contenant 570 facteurs du SUP, ce qui représente environ 60% des facteurs SUP humains connus. Puis nous avons mis au point une méthodologie permettant de réaliser un crible comparatif des interactions entre cette banque SUP et cinq PB2 provenant de souches de virus influenza A de virulence différentes chez l’homme : deux souches saisonnières circulant actuellement dans la population humaine (H1N1pdm09 et H3N2), deux souches hautement pathogènes chez l’homme (H7N9 et H1N1-1918) et une souche de laboratoire (H1N1-WSN). Cette première phase de cartographie a permis de sélectionner 42 facteurs du SUP interagissant avec au moins une des protéines PB2 étudiées. Par ailleurs, l’analyse des similarités de profils d’interaction PB2/UPS des souches étudiées a permis de mettre en évidence une corrélation avec le temps de circulation de chaque souche dans la population humaine. Nous avons ensuite caractérisé le rôle fonctionnel des partenaires de PB2 dans le cycle viral par des expériences de déplétion transitoire de l’expression des facteurs cellulaires par siARN, et validé 36 des 42 facteurs testés. La très grande quantité de facteurs identifiés impliqués dans le cycle viral démontre la qualité de la méthodologie développée pour l’identification de ces interacteurs. Parmi ces facteurs, nous avons étudié plus en détail le rôle de trois deubiquitinases (DUBs) dans l’infection. Nous avons montré que les DUBs sont impliquées dans les phases précoces et tardives du cycle viral. De plus, avec des collègues de Hong Kong nous avons mis en évidence que la DUB OTUB1 est impliquée dans la réponse cellulaire à l’infection produisant des cytokines, et probablement dans l’assemblage des nouveaux virions. Nous avons identifié que la DUB OTUD6A est également impliquée dans les phases tardives du cycle viral. A l’inverse PAN2 qui fait partie des complexes de poly-d’adénylation est impliqué dans les phases précoces. Nous poursuivons nos études afin d’élucider le rôle de ces DUBs dans l’infection par IAV. / An estimated 10%-20% of the world's population is affected each year by seasonal epidemic influenza, causing about 250,000 to 500,000 fatal cases. The pandemic risk reinforces the trait of influenza A virus (IAV) infection as a public health issue. The virus life cycle critically relies on its ability to manipulate the host proteome. Besides, the ubiquitin-proteasome system (UPS) is involved in many regulatory processes in mammalian cells by inducing protein degradation, mediating protein activation or shaping their sub-cellular localisation. Therefore, UPS is a prime target hijacked by viruses. Recent evidence indicates that an intricate regulatory network involving viral proteins and the cellular UPS is likely to contribute to viral replication and immune evasion of influenza A viruses. However, usurpation of the host UPS by IAV is far from being comprehensively deciphered. To gain better understanding, we assessed the interplay between the human UPS and the PB2 subunit of the influenza A virus polymerase through a global proteomic profiling approach. For that purpose, an UPS-dedicated library of 590 human cDNAs, comprising 63% of the whole human UPS, was constituted and characterised. In an initial screen, UPS factors were challenged using a high-throughput split luciferase assay for interaction with the PB2 protein from 5 influenza A strains of different pathogenicity in human. A total of 80 UPS factors emerged as potential PB2 partners, of which 42 were validated as high-confidence PB2 partners for at least one of the strains. Further comparison of interaction profiles of the 5 PB2 with the UPS by hierarchical clustering revealed an interaction dendrogram fitting with the circulation time in the human population.Functional importance of interactors was tested by siRNA-mediated knock down experiments using luciferase tagged recombinant IAV viruses. Depletion of 36 out of the 42 tested UPS factors showed an effect on the infection with all or a subset of IAV strains, underlying the strong functional output of the developed methodology. Among these factors three deubiquitinases (DUBs) were further studied to decipher their involvement in IAV viral cycle. We have shown that they are involved in early and late stage of the infection and began to draw their function in viral cycle. We demonstrated with our colleagues in Hong-Kong that OTUB1 is involved in the host cytokine response and most probably in virus assembly. OTUD6A was also shown to be implicated in late stages of the infection but we still don't know its exact role. Contrariwise, the inactive DUB PAN2, which is part of poly-deadenylation complexes, is implicated in early phase of IAV infection, but surprisingly apparently not through viral mRNA regulation. More work is on-going to precise by which mechanisms these DUBs are implicated in IAV infection.

Influenza A is a negative stranded RNA virus with a segmented genome. Once the virus infects a cell it must replicate its full length viral genomic RNA (vRNA) through a positive sense complementary intermediate RNA (cRNA) as well as transcribe viral messenger RNA (mRNA) using the vRNA as a template. The regulation of whether the viral polymerase replicates the genome by synthesizing cRNA, or produces mRNA in order to make viral protein involves, the viral nucleoprotein (NP). We tried to find the sequence residues of NP that directly interact with the viral polymerase. We mutated to alanine several residues on NP that are surface exposed on recently solved crystal structures as well as those thought to be oriented toward the viral polymerase complex in cryo-EM studies. As a first screen, we tested these mutants in a mini-genome assay where the NP stimulation of the viral polymerase can be studied in transfected cells. Through this screen we found that the NP mutants that hindered its ability to stimulate polymerase activity the most were located in a loop between two alpha helixes in the head domain of NP located at residues 203 to 209. Specifically, the NP single mutants of R204, W207, and R208 were inactive in the mini-genome assay. Using RT-PCR we found that the cRNA to vRNA step of replication is severely inhibited by these mutations. Immunoprecipitation using transfected cells showed that the NP mutants lost the ability to bind all three polymerase subunits. This indicates that this loss of polymerase binding may be the reason the NP mutant fails to stimulate polymerase activity. To make sure that this loss of polymerase stimulation was not due to altering other functions of NP we made sure that the protein had proper cellular localization, oligomerization, and RNA binding abilities. Using immuniflourescence we found that mutant NP localized to the nucleus just like wild type. In order to test RNA binding and oligomerization we tested NP purified from a baculovirus expressing system. Using fluorescence polarization we found that NP binds single stranded RNA with similar affinity to wild type. Using gel filtration we found that mutant NP forms oligomers just like wild type. Using covariation analysis of how different positions in an amino acid alignment change relative to each other we predicted possible binding sites between NP and the three polymerase subunits PA, PB1 and PB2. Due to more complete crystal structure data we focused on the PA-NP interaction and found that covariation aided in finding binding sequence residues on PA but not NP. Another outcome of developing the covariation method was developing a program to view broad primary structure changes in large sequence alignments. This method has been informative in evaluating how amino acid positions in influenza have changed over time, as well as what defines specific residues as belonging to human or avian viruses. / text

The structural characteristics associated with the binding of beneficial metals (i.e. - Mg2+, Zn2+ and Ca2+) to natural proteins has typically received more attention than competitive binding by toxic metals (e.g. – Pb2+, Hg2+, Cd2+, La3+, etc.). In this thesis, a statistical analysis of Pb2+-binding in crystallized protein structures indicates that Pb2+ does not bind preferentially with nitrogen, as generally assumed, but binds predominantly with oxygen, and to a lesser degree, sulfur. A comparison of Ca2+ and Pb2+ indicates that Pb2+ binds with a wider range of coordination numbers, with less formal change, and with less defined structure than Ca2+. The Pb2+ ion also appears to displace Ca2+ with little conformational stress in calcium binding proteins (CaBP’s). Experimental data from the binding of metals with engineered fluorescent proteins indicate that both Pb2+ and Gd3+ will occupy grafted calcium-binding sites with greater affinity than Ca2+, and strong evidence is presented to support the hypothesis that Pb2+ and Gd3+ will bind non-specifically on the protein surface. These results suggest that toxicity is associated with two binding mechanisms: displacement of the metal cofactor which disrupts protein function, and non-specific binding which maintains higher solubility of the metal.

Made available in DSpace on 2017-07-10T18:07:58Z (GMT). No. of bitstreams: 1 Andrea Colombo.pdf: 2540597 bytes, checksum: 5361b16fecab0f39c969122aa444a7d5 (MD5) Previous issue date: 2013-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this study the soybean hulls sorption potential in Cd2+ and Pb2+ in mono and binary component systems has been investigated at batch mode. In order to evaluate the effect of untreated and treated soybean hulls biomass, metal-bearing solution pH, sorption temperature, particle grain size, and agitation velocity on the improvement of the adsorption capacity of Cd2+ and Pb2+ cations, a series of adsorption preliminary tests, in duplicates, were performed. Regarding better sorption conditions, kinetic and equilibrium sorption experiments at batch mode were carried out for both mono and binary component systems. For preliminary and kinetic sorption experiments a series of 125 mL Erlenmeyer-type flask containing 300 mg dried soybean hulls in contact with 50 mL metal-bearing solutions at a 4 mequiv. L-1 concentration of Cd2+ and Pb2+ cations under controlled temperature and agitation velocity in a shaker. Regarding the equilibrium contact time, other sorption experiments, in duplicate, consisting of a series of mixture of 50 mL metal-bearing solutions and 50-1000 mg dried soybean hulls biomass were also performed under controlled temperature and agitation velocity in a shaker. In single component systems, a cation concentration of 4 mequiv. L-1 was tested, while in binary component systems three concentration combination in Cd2+-Pb2+bearing solutions were tested. After each sorption experiment, biosorbent was separated from metal solution by a 0.45 µm pore size membrane filtration system, performing then the determination of Cd and Pb concentrations in diluted liquid samples by flame atomic absorption spectrometry. Sorption tests regarding untreated soybean hulls biomass, pH 5 for Cd2+ and pH 4 for Pb2+, 30oC sorption temperature, a mixture of different particle grain sizes, and 100 rpm agitation velocity have showed the best results on the biosorbent performance to sequestrate Cd2+ and Pb2+ cations from metal-bearing solutions. The kinetic test data have shown that the equilibrium was achieved at contact times of 120 and 180 min for Cd2+ and Pb2+, respectively. Meanwhile, 55 and 64% concentration reductions for Cd2+ and Pb2+, respectively, were also achieved. Furthermore, the pseudo-second order kinetic model has fitted better the sorption kinetic data for both tested divalent cations. The equilibrium sorption data for mono-component systems have been well fitted by the Langmuir isotherm model, with maximum sorption capacity (qmax) of 0.5091 ± 0.0054 mequiv.g-1 and 0.6577 ± 0.0218 mequiv.g-1 for Cd2+ and Pb2+, respectively. Bicomponent equilibrium data were graphically represented by a 3D adsorption surface and modeled by a modified extended-to-multicomponent Langmuir-type isotherm, obtaining good fits. The results indicates that each component maximum capacity depends on the other species at the solution, inasmuch total adsorption capacity remains constant. Besides, a second component at the solution influences the isotherm curve shape. Thus, based on sorption results, untreated soybean hulls biomass is an alternative low-cost biosorbent to be applied in treatment systems of industrial effluents containing high concentration of Cd2+ and Pb2+ cations. / O objetivo deste trabalho foi avaliar o potencial da casca de soja no processo de biossorção, em sistema fechado batelada, das espécies metálicas cádmio (II) e chumbo (II) em soluções monocomponente e bicomponente. Foram realizados testes preliminares para verificar o efeito do tratamento no biossorvente, pH da solução, temperatura de sorção, granulometria do biossorvente e velocidade de agitação, na remoção dos íons metálicos. As melhores condições obtidas nos testes preliminares foram utilizadas nos testes cinéticos e de equilíbrio, monocomponente e bicomponente. Para os testes preliminares e cinéticos foram colocados, em frasco erlenmeyer de 125 mL, 300 mg de casca de soja em contato com 50 mL de solução com concentração de aproximadamente 4 mequiv.L-1 do íon metálico (cádmio ou chumbo). Para os testes de equilíbrio foram colocados, em frasco erlenmeyer de 125 mL, 50 a 1000 mg de biomassa com 50 mL de solução. No teste de equilíbrio monocomponente, a concentração inicial da solução foi em torno de 4 mequiv.L-1 do íon metálico (cádmio ou chumbo), e no bicomponente, as seguintes combinações de concentrações de cádmio e chumbo foram utilizadas: 4 - 4 mequiv L-1, 1 - 3 mequiv L-1; e 3 - 1 mequiv L-1. Todas as amostras foram mantidas com temperatura e agitação controladas por período pré-determinado. Em seguida, foram filtradas em membrana com tamanho dos poros de 0,45 µm, diluídas e analisadas em relação a concentração dos íons inicialmente presentes na solução por espectrofotometria de absorção atômica. Todos os testes foram realizados em duplicata. Os resultados dos testes preliminares mostraram que as melhores condições para biossorção dos íons cádmio e chumbo foram casca de soja sem tratamento (in natura), pH 4, temperatura de adsorção de 30°C, mistura granulométrica do biossorvente e velocidade de agitação 100 rpm. Nos testes cinéticos o tempo de equilíbrio foi de 120 min para o cádmio, com taxa de remoção de 55%, e de 180 min para o chumbo, com uma taxa de remoção de 64%. O modelo cinético de pseudosegunda ordem foi o que melhor representou os dados experimentais para ambos os íons metálicos. Aos dados de equilíbrio, o modelo que melhor se ajustou foi o de Langmuir cujos parâmetros q_max e b para os íons de Cd (pH 5) e Pb (pH 4) foram: 0,50910 ± 0,0054 mequiv.g-1 e 1,16236 ± 0,03242 L.mequiv-1 e 0,65773 ± 0,02181 mequiv.g-1 e 0,74735 ± 0,09479 L.mequiv-1. Os dados de equilíbrio, bicomponente, foram representados graficamente por meio da superfície de adsorção e modelados pelo modelo de Langmuir estendido modificado, obtendo-se bom ajuste. Os resultados mostraram que a máxima capacidade de cada espécie metálica é um parâmetro dependente de outra espécie da mistura, enquanto que a capacidade de adsorção total assume um valor constante. Além disso, a presença de outro metal em solução afetou a forma da curva da isoterma. Assim, pelos resultados obtidos pode-se afirmar que a casca de soja possui grande potencial para o tratamento de efluentes contendo cádmio e chumbo.