No description available.

<p>To investigate mechanisms involved in virus-host interactions, global changes in host gene expression were examined during infection with adenovirus type 2 (Ad2) using cDNA microarray technology. </p><p>In paper I and II, transcriptional changes in HeLa cells were investigated during the early and late phase of infection, respectively. A limited number of genes, mainly implicated in cell growth and antiviral defence, were found to be differentially expressed in the early phase, whereas modulation of host cell gene expression during the late phase was augmented and mainly focused on growth inhibition and cell architecture. </p><p>The experimental set-up was then redesigned to follow transcriptional regulatory events in growth synchronised, human primary lung fibroblasts. The immediate response of the host cell within two hours of infection was investigated in paper III, revealing a transient induction of a small number of cellular alert genes. This was followed by an expanded time course presented in paper IV, which included gene expression profiling at eight consecutive time points throughout the infectious cycle. The results indicated that specific sets of cellular genes were targeted at different stages of the infection, and four distinct periods were identified. </p><p>In summary, the studies presented in this thesis demonstrate that adenovirus interferes with many cellular processes during the progression of infection to optimize the cellular environment for viral replication. These include cell cycle control, cell growth and growth inhibition, as well as DNA, RNA and protein metabolism. However, a transient induction of cellular genes involved in immune response and growth inhibition was observed before the onset of viral gene expression. During the very late stages of infection, the expression of a large number of genes involved in maintaining the cell structure was down-regulated, presumably to facilitate the spread of progeny virus.</p>

To investigate mechanisms involved in virus-host interactions, global changes in host gene expression were examined during infection with adenovirus type 2 (Ad2) using cDNA microarray technology. In paper I and II, transcriptional changes in HeLa cells were investigated during the early and late phase of infection, respectively. A limited number of genes, mainly implicated in cell growth and antiviral defence, were found to be differentially expressed in the early phase, whereas modulation of host cell gene expression during the late phase was augmented and mainly focused on growth inhibition and cell architecture. The experimental set-up was then redesigned to follow transcriptional regulatory events in growth synchronised, human primary lung fibroblasts. The immediate response of the host cell within two hours of infection was investigated in paper III, revealing a transient induction of a small number of cellular alert genes. This was followed by an expanded time course presented in paper IV, which included gene expression profiling at eight consecutive time points throughout the infectious cycle. The results indicated that specific sets of cellular genes were targeted at different stages of the infection, and four distinct periods were identified. In summary, the studies presented in this thesis demonstrate that adenovirus interferes with many cellular processes during the progression of infection to optimize the cellular environment for viral replication. These include cell cycle control, cell growth and growth inhibition, as well as DNA, RNA and protein metabolism. However, a transient induction of cellular genes involved in immune response and growth inhibition was observed before the onset of viral gene expression. During the very late stages of infection, the expression of a large number of genes involved in maintaining the cell structure was down-regulated, presumably to facilitate the spread of progeny virus.

No description available.

Les microorganismes du domaine du vivant Archaea sont très divers sur le plan biologique et sont présents dans de nombreux types d'écosystèmes. Ils sont majoritaires dans les environnements dits extrêmes. Parmi les virus d'archées, ceux infectant les espèces d'un phylum majeur des archées, Crenarchaeota, constitué d'hyperthermophiles, forment un groupe exceptionnel. En effet, leurs morphotypes sont uniques, variés, et complexes. Le contenu de leur génome est également unique. Enfin, la plupart de ces virus se maintiennent dans la cellule hôte en état porteur, une relation chronique qui permet un équilibre entre production de virions et division cellulaire. J'ai d'abord démontré que le virus de crenarchée Sulfolobus islandicus rod-shaped virus 2 est un virus virulent, et non chronique comme il avait été suggéré. Un mécanisme de lyse unique a été découvert. La paroi cellulaire est modifiée en plusieurs points, avec l'apparition de structures pyramidales saillantes. Celles-ci s'ouvrent en fin de cycle infectieux, permettant aux virions, assemblés auparavant dans le cytoplasme, de quitter la cellule. Puis j'ai travaillé sur des échantillons de sources géothermiques de la péninsule de Kamchatka (Russie) et contribué à l'isolement et la caractérisation d'un virus de morphotype filamenteux. Des protéines structurales supplémentaires ont ainsi été identifiées. Enfin, des mots courts exceptionnels ont été identifiés dans un grand nombre de génomes d'archées et de leurs éléments extra-chromosomiques. Ce sont potentiellement des motifs fonctionnels non-codants, impliqués dans des mécanismes biologiques importants. Typiquement, les motifs palindromiques sont évités dans les génomes / The microorganisms from the Archaea domain are very diverse at the biological level and they are present in many types of ecosystems. They are dominant in the so-called extreme environments. Among their viruses, those infecting species of the Crenarchaeota phylum, a major archaeal phylum comprising hyperthermophiles, form an exceptional group. Indeed, their morphotypes are unique, diverse, and complex. Their genome content is also unique. Finally, most of these viruses persist in the host cell in a carrier state, a chronic relationship allowing an equilibrium between virion production and cell division. I first proved that Sulfolobus islandicus rod-shaped virus 2 is a virulent virus, and not chronic, as had previously been suggested. A unique lysis mechanism was discovered. The cell wall is modified in several locations, with the appearance of pyramidal prominent structures. Those burst open at the end of the infection cycle, allowing the virions, previously assembled in the cytoplasm, to leave the cell. Then, I worked on environmental samples from geothermal springs of the Kamchatka peninsula (Russia) and contributed to the isolation and characterization of a virus of filamentous morphotype. Additional structural proteins were in particular identified. Finally, short exceptional words were identified in a great number of genomes from archaea and their extra-chromosomal elements. These are potentially functional non-coding motifs involved in important biological mechanisms. Typically, palindromic motifs are avoided in the genomes

The abundant cellular protein Cyclophilin A (CypA) was found to bind to HIV-1 capsid (CA) in 1993. Since that time, several complementary methods, including disruption of the binding interface by cyclosporine A, CA mutants, and CypA mutants, have been used to demonstrate that CypA acts within human target cells to promote HIV-1 infection. In contrast, in cells from non-human primates, CypA in target cells decreases HIV-1 infectivity, and it does so by promoting TRIM5α-mediated restriction. Using human cancer cell lines and the genetic methods available at the time, attempts to obtain evidence that CypA inhibits HIV-1 restriction by the human TRIM5α ortholog, let alone that human TRIM5α restricts HIV-1, were unsuccessful. Here we revisit the question of the mechanism by which CypA increases HIV-1 infectivity by exploiting lentiviral vectors optimized for primary human blood cells that serve as HIV-1 targets. Disruption of CA−CypA interaction is demonstrated to render HIV-1 vulnerable to endogenous human TRIM5α-mediated recognition and restriction, which occur prior to completion of reverse transcription. Identical findings were acquired with single-cycle vectors or with replication-competent viruses. Consistently, a previously identified, cyclosporine-resistant CA mutation A92E is also shown to confer resistance against restriction by human TRIM5α. Therefore, the results presented in this thesis reveal that HIV-1 exploits a host protein CypA bound to its CA to evade potent restriction by human TRIM5α. This finding not only answers a long-standing question regarding the role of CypA in HIV-1 infection, but also may reinvigorate the development of CypA inhibitors for treatment of HIV-1.

Peroxisome biogenesis is complex, involving a diverse array of intracellular pathways and mechanisms that mediate their biogenesis and cellular functions. Relevant to our understanding of peroxisome biogenesis is the utilization of peroxisomal membranes for viral genome replication as observed in plant cells infected by several members of the Tombusviridae family of positive-strand RNA viruses. Tomato Bushy Stunt Virus (TBSV), for instance, usurps an array of host factors that facilitate the transformation of peroxisomes into peroxisomal multivesicular bodies (pMVB) the sites of viral RNA replication. In this study, pMVB topology and biogenesis was investigated using transmission electron and epifluorescence microscopy of tobacco and wildtype or mutant budding yeast that were transformed with TBSV replicase proteins and a defective interfering viral RNA. Overall, the results suggest that host-virus interactions specifically associated with Endosomal Sorting Complex Required for Transport (ESCRT) and lipid metabolism are involved in TBSV replication and pMVB biogenesis.

No description available.

The egress pathway of herpes simplex virus-1 (HSV-1) is a complicated process mediated by co-ordinated activity of several virus glycoproteins. The virions are first assembled and enveloped at trans-Golgi-network (TGN) or endosome membranes and then travel through a guided pathway that is directed towards the cell adherent points for secretion. Once secreted the vast majority of virions remain associated with the extracellular membrane of cells and very few free virions are released into the culture medium (< 1%). The mechanisms that mediate both the targeted secretion of newly assembled virions at cell contact points and post-secretion attachment of virions with the extracellular surface of cells are poorly understood, and were the topics of this research. In this thesis, an HSV-1 passage mutant of increased virion secretion phenotype had been studied. Genome sequencing of the mutant virus identified mutations in three viral envelope proteins. Study of recombinant viruses that were constructed based on those three mutations revealed that a single amino acid change in glycoprotein I (gI) of glycine to arginine at residue 39 is responsible for the increased release of virus. The result suggests the principal effect of this mutation is to modify the secretory pathway used by virions during their release from infected cells. Data also suggests a role of gC in the attachment of virions to the extracellular surface of cells after egress. In the context of HSV-1 envelopment and egress glycoprotein E (gE), which forms a heterodimeric complex with gI (gE/gI), is known to be important. The gE/gI complex has been shown to interact with many tegument proteins and have a redundant role in secondary envelopment. The gE/gI complex has been also proposed to colocalise with various cellular components and sort the nascent virions to cell contact points. However, there is little understanding of the cellular proteins that gE/gI interact with, or the mechanisms that mediate targeted secretion of virions. This research has identified a novel interactome of gE/gI by mass-spectrometric analysis utilising stable isotope labelling with amino acids in cell culture (SILAC) medium. Among the cellular interactome obtained, Nipsnap1 was validated by co-precipitation assays from both infected and transfected cells, and furthermore using cell free systems, suggesting gE and Nipsnap1 directly interact. Nipsnap1 and its homologue Nipsnap2 have been proposed to contribute in vesicle transport and membrane fusion in cells. Using CRISPR-Cas9 technology these proteins were knocked out in a keratinocyte cell line (HaCaT) to investigate their role in HSV-1 egress. However, little or no effect on HSV-1 egress could be observed upon loss of either or both of these proteins suggesting the biological significance of gE-Nipsnap1 interaction may not be directly linked to any egress function of gE/gI. Two further interesting 'hits' from the gE/gI interactome were interferon-induced transmembrane protein type-2 (IFITM2), a virus restriction factor, and Myoferlin that has a putative role in endocytic vesicle recycling. This study could validate gE-Myoferlin interaction and co-localisation in infected or transfected cells however, functional significance of this interaction remains to be determined. Overall, the research of this thesis has provided a better understanding of the role of the gE/gI complex in HSV-1 egress and investigated the role of some interesting cellular proteins in the context of virion egress.

La réplication et l’assemblage du virus de l’hépatite C (VHC) sont régulés finement dans le temps et l’espace par les interactions protéiques entre le virus avec l’hôte. La compréhension de la biologie du virus ainsi que sa pathogénicité passe par les connaissances relatives aux interactions virus/hôte. Afin d’identifier ces interactions, nous avons exploité une approche d’immunoprécipitation (IP) couplée à une détection par spectrométrie de masse (MS), pour ensuite évaluer le rôle des protéines identifiées dans le cycle viral par une technique de silençage génique. Les protéines virales Core, NS2, NS3/4A, NS4B, NS5A et NS5B ont été exprimées individuellement dans les cellules humaines 293T et immunoprécipitées afin d’isoler des complexes protéiques qui ont été soumis à l’analyse MS. Ainsi, 98 protéines de l’hôte ont été identifiées avec un enrichissement significatif et illustrant une spécificité d’interaction. L’enrichissement de protéines connues dans la littérature a démontré la force de l’approche, ainsi que la validation de 6 nouvelles interactions virus/hôte. Enfin, le rôle de ces interactants sur la réplication virale a été évalué dans un criblage génomique par ARN interférant (ARNi). Deux systèmes rapporteurs de la réplication virale ont été utilisés : le système de réplicon sous-génomique (Huh7-Con1-Fluc) et le système infectieux (J6/JFH-1/p7Rluc2a), ainsi qu’un essai de toxicité cellulaire (Alamar Blue). Parmi les protéines de l’hôte interagissant avec le VHC, 28 protéines ont démontré un effet significatif sans effet de toxicité cellulaire, suggérant fortement un rôle dans la réplication du VHC. Globalement, l’étude a mené à l’identification de nouvelles interactions virus/hôte et l’identification de nouvelles cibles thérapeutiques potentielles. / Hepatitis C virus (HCV) replication and assembly are tightly regulated in time and space within the cell, most likely due to protein interactions between virus and host. In order to better understand HCV biology and its pathogenesis, there is a need to unravel virus/host interaction network. We extended our knowledge of virus/host interactions by the identification of cellular proteins associated to HCV proteins using an immunoprecipitation (IP) technique coupled to mass spectrometry (MS), and further evaluate the role of retrieved interactors using gene knockdown. FLAG-tagged viral proteins Core, NS2, NS3/4A, NS4B, NS5A and NS5B have been expressed individually in 293T human cells, and immunoprecipitated protein complexes have been submitted to MS analysis for identification of host proteins. In this study, 98 proteins were significantly enriched and showed specific interaction to a viral protein. Retrieval of previously characterized interacting proteins proved the strength of the method. Six newly identified interactors by MS were individually confirmed using IP of viral proteins. We evaluated the role of identified interactors in HCV replication by performing a functional lentivirus-based RNA interference (RNAi) screen. Two reporter systems were used: the sub- genomic replicon (Huh7-Con1-Fluc) and a full length infectious clone (J6/JFH-1/p7Rluc2a), as well as the cellular toxicity assay Alamar blue. Of the identified host interactors, 28 proteins showed a significant effect on HCV replication upon gene knockdown and without cellular toxicity. Overall, the study led to the identification of novel virus/host interactions essential in HCV life cycle and provides novel potential drug targets.