Made available in DSpace on 2018-06-27T18:56:08Z (GMT). No. of bitstreams: 2 Adrianne Del Fabro Ceccim.pdf: 4991781 bytes, checksum: d1128fe71a2a03c2beaa42a7847dfedb (MD5) Adrianne Del Fabro Ceccim.pdf.jpg: 3245 bytes, checksum: 583d4670e00da5b265b326517cddf626 (MD5) Previous issue date: 2011-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Thousands of people die annually due to infections caused by some kind of virus, despite the large investment on development of technologies that improve the diagnostics and prognostics of virus diseases. However, there is a lack of methods for diagnosis that are more accurate, low cost and that handle problems such as the immunologic window, the bounds of different infection phases, as well as the low amount of virions. The Hsps 70 are molecular chaperones that are expressed in various situations of stress or heat, forming a highly preserved immunologic system. They are highly expressed in different kind of tumors and in some in almost all viral infections. In the same way, its co-chaperona, the heat chock protein 70 linked to protein 1 (HspBP1) is involved in tumors processes, such as the lung cancer and the neuroblastoma. Recently, it was identified as a marker for the prognostic of breast cancer. Despite having studies related to the expression of HspBP1 on cancers and on its prognostics, the studies of its expression in viral infections are rare. Due to the fact that this protein can be used as an indicator of cellular metabolic alteration and that the viral infection will induce the cellular stress, the modification of its expression during the viral cycle can predict a higher or lower replicative index. This property can be used as a tool in the viral diagnostic. The mainly objective of this study was to determine the pattern of expression of protein HspBP1 in cells that hosts HSV-1 (vero cells) in vitro using the Western-blotting and determine increasing or reduction on its expression. In addition, the levels of HspBP1 and antibodies agains HspBP1 in the serum of HIV infected people were analized by ELISA. The results in vitro showed that after the viral infection there was no different in the pattern of expression of the total protein, when analyzed by SDS-PAGE. However, using the Western-Blotting the expression of HspBP1 increased in the 48 hours after the infection, comparing with non-infected Vero cells. Seventy-two hours after infection the expression decreased. The results of HspBP1 protein by ELISA showed a significant increasing of this protein in the HIV infected group that also presented a high viral load and low number of T CD4+ lymphocytes, when compared with the groups with an undetected load of HIV infected and uninfected. Regarding the research of anti-HspBP1 the results showed an increase in serum protein from HIV infected people. In conclusion these results suggest that the expression of HspBP1 is increased in HIV infected people and it is followed by an increasing in antibody production against HspBP1 and decreasing in T CD4+ lymphocytes for high viral load. / Milhares de pessoas morrem anualmente por infecções causadas por algum tipo de vírus, mesmo que, nos últimos anos, se invista muito no desenvolvimento de tecnologias para aprimorar o diagnóstico e prognóstico de viroses. Entretanto, ainda existe uma carência de métodos diagnósticos mais sensíveis, de baixo custo, que contornem problemas como a janela imunológica, os limites de detecção em determinadas fases da infecção, assim como a baixa quantidade de vírions. As Hsps 70 são chaperonas moleculares que se expressam em várias situações de estresse e ou calor, constituindo um sistema genético altamente conservado, sendo altamente expressas em diversos tipos de tumores e em certos tipos de viroses. Do mesmo modo, a sua co-chaperona, a proteína de choque térmico 70 ligada à proteína 1 (HspBP1), está envolvida em processos tumorais, como no câncer de pulmão e no neuroblastoma, e recentemente, foi identificada como um marcador de prognóstico no câncer de mama. Embora existam estudos relacionando à expressão da HspBP1 no câncer e o prognóstico deste, os estudos de sua expressão em infecções virais são raros. Em razão dessa proteína poder ser utilizada como um indicador de alteração metabólica celular, e a infecção viral induzir estresse celular, a alteração de sua expressão durante o ciclo viral pode predizer um maior ou menor índice replicativo. Assim, essa propriedade pode ser utilizada como ferramenta no diagnóstico viral. Dessa forma, este trabalho teve como objetivo determinar o padrão de expressão da proteína HspBP1 em células hospedeiras do vírus HSV-1 (células Vero) in vitro através da técnica de Western-blotting, avaliando um provável aumento ou diminuição da sua expressão. Além disso, quantificar a proteína HspBP1 e os anticorpos IgG anti-HspBP1 no soro de indivíduos HIV positivos através da técnica de ELISA. Os resultados demonstraram que após a infecção viral não houve diferença no padrão de expressão de proteína total, quando analisados por SDS-PAGE. No entanto, pela técnica de Western-Blotting, a expressão de HspBP1 apresentou aumento nas 48 horas após a infecção em comparação com células Vero não infectadas. Setenta e duas horas após a infecção, a expressão diminuiu. Os resultados da pesquisa da proteína HspBP1 pelo teste ELISA mostraram que houve um aumento significativo dessa proteína no grupo dos indivíduos infectados pelo HIV apresentando carga viral alta e contagem de linfócitos T CD4+ baixos quando comparado com os grupos dos infectados pelo HIV com carga viral indetectável e com os não infectados. Em relação a pesquisa de anticorpos IgG anti-HspBP1 os resultados mostraram que houve um aumento da proteína em indivíduos infectados pelo HIV. Assim, esses resultados sugerem que a expressão HspBP1 está aumentada em indivíduos infectados pelo HIV, seguidos por um aumento na produção de anticorpos contra a HspBP1 e redução dos linfóctios T CD4+ para uma carga viral elevada.

Brome mosaic virus (BMV) belongs to a viral class called positive-strand RNA [(+)RNA] viruses. This is the largest class of viruses and includes numerous important pathogens. BMV infects monocotyledonous plants and its replication can be recapitulated in the baker's yeast (Saccharomyces cerevisiae) under laboratory conditions to use yeast as an experimental model system. BMV generally does not infect dicotyledonous plants including the model plant Arabidopsis thaliana. One important shared feature of (+)RNA viruses is that they all make use of host proteins to aid in their own viral replication. In particular, (+)RNA viruses use host intracellular membranes for their replication and lipid composition of these membranes is crucial for viral replication. I show here that BMV replication protein 1a causes redistribution of host Lam5 (Lipid transfer protein Anchored at a Membrane contact site 5) and that Lam5 is necessary for BMV replication in yeast. Furthermore, in the absence of Lam5, BMV 1a affects the distribution of lipid droplets throughout yeast cells. Host factors also play critical roles in defense against viruses. Although wild-type Arabidopsis is not a host for BMV, the Arabidopsis cpr5 (Constitutive expression of Pathogenesis-Related genes 5) mutant can support systemic infection of BMV. I performed screens in Arabidopsis and have identified four genes that contribute to defense against BMV. These include two RNA-binding proteins, a lectin superfamily protein, and an alternative oxidase. My results also contribute to the growing evidence that reactive oxygen species play a key role in BMV replication. In summary, my work provided new insights into BMV replication in hosts and plant defense against BMV infection. The information gained from these projects aids in our understanding of (+)RNA virus biology in general and may identify targets for developing broad-spectrum antiviral strategies. / Doctor of Philosophy / Viruses are important pathogens that can cause devastating diseases not only in humans, but also in animals and plants. It is important to study viruses and their interactions with their hosts to develop antiviral drugs and engineer plant resistance to viruses. Positive-strand RNA viruses are the largest class of viruses and are responsible for numerous human, animal, and plant diseases. Brome mosaic virus (BMV) belongs to this class of viruses and has a simple genome organization. Furthermore, the baker's yeast can support BMV replication. BMV serves as a model to study (+)RNA virus replication and virus-host interactions. I show here that BMV depends on the lipid transport protein Lam5 for proper replication in yeast. Furthermore, although much is known about BMV replication in yeast, more information is needed on BMV replication in plants. To aid in this goal, I performed screens in the model plant Arabidopsis thaliana to identify Arabidopsis mutants that could allow for systemic BMV infection. This project has uncovered four new genes that contribute to defense against BMV. This research aids in our understanding of how BMV replication works and also how plants defend themselves against viruses. This work is important for understanding of the biology of (+)RNA viruses and the plant immune system in general.

+ssRNA viruses after entering the cell develop platforms for RNA replication called replication organelles. Due to the activity of phosphatidylinositol 4-kinases is in these areas a higher concentration of PI4P, which establishes suitable binding environment for the viral polymerase 3DPOL . One of these kinases is PI4KB, which is recruited to the membrane by the ACBD3 protein, which is itself recruited by giantin. Some kobuviruses and enteroviruses from the Picornaviridae family use their 3A protein to displace ACBD3 protein from the complex with giantin and transfer it from Golgi aparathus to the replication organelles. Here, PI4KB binds to ACBD3 protein and synthesizes PI4P. Recently, two proteins - TBC1D22A and TBC1D22B - were discovered to bind to the same area of ACBD3 protein as PI4KB. The goal of this project was verification of this interaction and its subsequent characterization (e.g. dissociation constant measurements). My goal was to crystallize complexes of these interaction partners and to solve three-dimensional structure. Our results suggest, that interaction of ACBD3 protein with peptides derived from TBC1D22A and TBC1D22B proteins is much lower compared to interaction between ACBD3 protein and PI4KB. I successfully prepared crystals, however, they diffracted poorly, not allowing us to solve...

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. Despite decades of research, it is still unclear how these viruses establish a lifelong, asymptomatic infection in their rodent hosts while infection of humans often results in severe disease. Unable to enter a state of bona fide latency, the transcription and replication of the viral genomic RNA is likely highly regulated in time and subcellular space. Moreover, we hypothesize that the viral nucleoprotein (NP), responsible for the encapsidation of the viral RNA and the most highly expressed viral gene product, plays a key role in the regulation of the viral gene expression program. Further, exploring host-virus interactions may elucidate the basic aspects of arenavirus biology and how they cause such severe disease in humans. To explore these questions in greater detail, this dissertation has pursued three main avenues. First, to better understand lymphocytic choriomeningitis mammarenavirus (LCMV) genome replication and transcription at the single-cell level, we established a high-throughput, single-molecule (sm)FISH image acquisition and analysis pipeline and followed viral RNA species from viral entry through the late stages of persistent infection in vitro. This work provided support for a cyclical model of persistence where individual cells are initially transiently infected, clear active infection, and become re-infected from neighboring reservoir cells within the population. Second, we used FISH to visualize viral genomic RNA to describe the subcellular sites where LCMV RNAs localize during infection. We observed that, viral RNA concentrates in large subcellular structures located near the cellular microtubule organizing center and colocalizes with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane bound organelle as a site for the pre-assembly of viral components including genomic RNA and viral glycoprotein prior to their transport to the plasma membrane where new particles will bud. Last, we used mass spectrometry to identify human proteins that interact with the NPs of LCMV and Junín mammareanavirus (JUNV) strain Candid #1. We provided a detailed map of the host machinery engaged by arenavirus NPs, and in particular, showed that NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. We demonstrated that JUNV antagonizes the antiviral activity of PKR completely, effectively abrogating the antiviral activity of this surveillance pathway. In sum, the work composing this dissertation has given us fresh insight into how arenaviruses establish and maintain persistence; the nature of the subcellular site where viral genomic RNA is transcribed, replicated, and assembled with other viral components; and a global view of the cellular machinery hijacked by the viral nucleoprotein. This work improves our basic understanding of the arenavirus life cycle and may suggest novel antiviral therapeutic targets that could be exploited in the future.

GBF1 a émergé autant que facteur cellulaire nécessaire pour la réplication de plusieurs virus à ARN. Au cours de l’infection par le virus de l’hépatite C (VHC), GBF1 est essentiel pour les étapes précoces de la réplication, bien qu’il soit dispensable lorsque celle-ci est établie. Afin de mieux comprendre la fonction de GBF1 dans la régulation de l'infection par le VHC, nous avons tenté d’explorer les interactions entre GBF1 et les protéines du VHC. Ainsi, grâce à l’approche du double hybride en levure et par co-immunoprécipitation et par PLA (proximity ligation assay), nous avons pu montrer que NS3 interagit avec GBF1. De plus, NS3 semble interférer avec la localisation subcellulaire de GBF1 dans des cellules exprimant NS3. Cette interaction a été retrouvée entre le domaine protéase de NS3 et Sec7, le domaine catalytique de GBF1. Un crible sur des mutations altérant l’interaction GBF1-NS3, par double hybride en levure, a permis révéler un mutant NS3 (N77D de la souche Con1) qui est non-réplicatif malgré une activité protéase bien conservée. De plus, le résidu muté est exposé à la surface, ce qui suggère qu’il pourrait appartenir à la zone d’interaction de NS3 avec GBF1. La mutation correspondante dans la souche JFH1 produit le même phénotype que la souche Con1 du VHC. L’ensemble des résultats révèlent l’existence d’une interaction entre GBF1 et NS3 et suggèrent qu’une altération de cette interaction est délétère pour la réplication du VHC. / GBF1 has emerged as a host factor required for the replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of replication, but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, in co-immunoprecipitation and in proximity ligation assays, and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A yeast two-hybrid screen for mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is non-replicative despite conserved protease activity. The mutated residue is exposed at the surface of NS3, suggesting it could be part of the domain of NS3 that interacts with GBF1. The corresponding mutation in JFH-1 strain (S77D) produces the same phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV replication.

Le virus de la maladie de Marek (MDV) est un alphaherpesvirus à l’origine du développement de lymphomes chez les volailles. A ce jour, l’origine du développement des tumeurs induites par le MDV est encore peu connue malgré l’identification de plusieurs oncoprotéines virales. Au vu de l’implication des dommages à l’ADN dans la pathogenèse de plusieurs virus notamment les herpesvirus, mon projet de thèse avait pour objectif de déterminer l’impact de l’apparition de lésions d’ADN sur le cycle viral du MDV. Nous avons montré que l’infection cytolytique de MDV s’accompagne d’une accumulation de lésions dans l’ADN cellulaire de lymphocytes et cellules fibroblastiques de poulet. La phase de latente de l’infection MDV n’affecte pas l’intégrité de l’ADN des lymphocytes alors que la réactivation du virus conduit à l’apparition de lésions d’ADN. De plus, en utilisant une approche originale in vivo, nous avons confirmé le rôle essentiel de la protéine virale VP22 dans l’induction de ces dommages. Nous avons pu établir que l’induction des dommages à l’ADN au cours de l’infection et/ou la réponse cellulaire engendrée sont non seulement favorables à la réplication du virus mais également que l’apparition de ces lésions est étroitement liée au pouvoir oncogène du MDV. / Marek’s disease virus (MDV) is an alphaherpesvirus responsible of T lymphoma in chickens. Mechanisms leading to cellular transformation mediated by MDV are still incompletely understood. DNA damage and the associated cellular response participate actively in the life cycle of viruses, especially herpesviruses. Here, we aimed at deciphering the role of DNA damages in MDV pathogenesis. We show that MDV lytic infection leads to DNA lesions in lymphocytes and fibroblasts of chickens. Moreover, we demonstrated that MDV latently-infected lymphocytes exhibits undamaged DNA whereas MDV reactivation leads to an onset of DNA lesions. Also, using an original in vivo approach, we objectified the role of VP22 on DNA damages induction. Finally, we established that DNA damage and/or the associated DNA damage response are not only benefic to MDV replication but also that the DNA lesions onset might participate to MDV oncogenicity.