Increasing number of observations indicate that RNA interference (RNAi) may be used by mammalian cells to counteract virus infection as a natural innate immunity. The human immunodeficiency virus (HIV-1) TAR RNA binding protein (TRBP) interacts with Dicer and is a component of the RNA-induced silencing complex (RISC). In humans, while Dicer alone can process double-stranded (ds) RNAs and pre-micro (mi)RNAs efficiently, the recruitment of the short interfering (si)RNA/miRNAs to Ago2 requires TRBP. TRBP is a dsRNA binding protein that increases translation from HIV TAR containing RNAs by a direct binding to the structured TAR RNAs and by inhibiting the protein kinase PKR. The relationship between its activity on HIV gene expression and on RNAi has not been studied. Using the two-hybrid method we identified the specific region of interaction in the C-terminal part of TRBP and in the N-terminal part of Dicer. This interaction between both endogenous proteins was further confirmed by immunoprecipitation and colocalization in human cells. TRBP's requirement in the RNAi pathway was shown by an assay in HeLa, Astrocytes and Tarbp2 -/- cells using a green fluorescent protein-expressing plasmid and either short hairpin (sh)- or mi-RNAs against GFP. RNAi is decreased in Tarbp2 -/- mouse cells and Astrocytes. Results using a dual luciferase assay show that HIV-1 disrupts the RNAi mechanism in mammalian cells and that HIV-1 products Tat protein, TAR and RRE RNAs mediate this suppression. In addition, we show that HIV infection has no effect on the interaction between TRBP, Dicer and Ago2 in human cells. Even though the RNAi pathway is functional in human cells, HIV has found the way to repress this mechanism. Whether HIV-1 is able to disrupt the function of RISC to avoid an innate immunity response or whether it co-opts the RNAi pathway for its benefit remains to be elucidated. / Un grand nombre d'observations indiquent que l'interférence à ARN (RNAi) peut être utilisée comme une réponse naturelle innée dans des cellules de mammifère pour contrer l'infection virale. La protéine liant l'ARN TAR du virus d'immunodéficience humaine (VIH) (TRBP) est une composante du complexe de mise en silence induit par l'ARN (RISC), tel que démontré par son interaction avec Dicer. Bien que Dicer seul puisse cliver les ARNs double brin (db) et les pre-micro (mi)RNAs efficacement, le recrutement de « short interfering » (si)RNA/miRNAs à Ago2 a besoin de TRBP. TRBP, une protéine capable de lier l'ARN db, peut accroître la traduction du VIH contenant des ARNs TAR en se liant directement aux ARNs TAR structurés et en inhibant la protéine kinase PKR. La relation entre l'activité sur l'expression des gènes du VIH et la RNAi n'a pas encore été étudiée. En utilisant la méthode du double-hybride, nous avons identifié les régions spécifiques d'interaction dans la partie C-terminale de TRBP et dans la partie N-terminale de Dicer. On a confirmé l'interaction entre les deux protéines endogènes par immunoprecipitation et colocalisation dans des cellules humaines. On a montré que la voie de RNAi a besoin de TRBP, par un essai fonctionnel dans des cellules HeLa, des Astrocytes et des cellules de souris Tarbp2 -/- en utilisant des plasmides codant pour la fluorescence verte (GFP) et des short hairpin (sh)- ou miRNAs contre la GFP. La RNAi diminue dans des cellules de souris Tarbp2 -/-. Un essai de double luciférase démontre que le VIH supprime le mécanisme de RNAi dans des cellules humaines ainsi par l'intermédiaire de sa protéine Tat et de ses ARNs TAR et RRE. De plus, on démontre que l'infection par le VIH n'a pas d'effet sur l'interaction entre TRBP, Dicer et Ago2 dans les cellules humaines. Donc, même si la voie de RNAi est fonctionnelle chez l'humain, le VIH a trouvé des moyens pour supprimer ce mécanisme. Que le VI

No description available.

Neisseria meningitidis (SD1C) when grown in an iron-limited medium at low pH exhibited a dramatic increase in virulence for the mouse. This enhanced virulence correlated with a specific colonial type, and altered outer membrane protein profile, the expression of an uptake sytem for iron from human transferrin, and an increased resistance to phagocytosis by mouse polymorphonuclear leukocytes. The outer membrane of cells grown at pH 6.6 contained a 69,000 molecular weight protein (protein III) that was exposed on the cell surface. The outer membrane of iron-limited cells grown at pH 7.2 also exhibited protein III, but cells grown at pH 7.2 with sufficient iron possessed only minute quantities of this protein. Only iron-limited cells actively accumulated iron from human transferrin. Iron-limited cells initially took up ('59)Fe from transferrin at a similar rate, irrespective of their growth pH, but after 60 minutes cells grown at neutral pH accumulated less than half the ('59)Fe of cells from pH 6.6 cultures. At 24 hours, N. meningitidis (M2092) produced small and large colonies in which the cells elaborated predominantly thin (2.5 nm) and thick (4.5 nm) pili, respectively. The piliated meningococci possessed a sizeable intracellular pili(n) pool and, although in vitro pili reassembly was energy-independent, the in vivo assembly of pili on the meningococcal surface required a functional respiratory chain. Meningococci normally piliated at pH 7.2 were nonpiliated at pH 6.6, whereas nonpiliated meningococci at pH 7.2 remained nonpiliated at low pH. The effect of simulated in vivo growth conditions on meningococcal phenotype and virulence are discussed.

Bacterial invasion of vertebrates triggers a marked reduction in the levels of iron associated with the plasma transferrin (Tf) pool. This hypoferremic response has been regarded as a host attempt to withhold essential iron from the invading pathogen. The exact nature of the mechanisms involved remains obscure. / The plasma Tf iron pool was studied with radiolabeled mouse Tf and its kinetics were determined in normal and infected mice. The plasma Tf iron pool of normal mice was dynamic with a half-life of 0.7 h. Iron left the plasma pool, entered the bone marrow, and was incorporated into erythrocytes. Studies with mice infected with Neisseria meningitidis revealed similar kinetics including no redistribution of iron within the various iron pools. Iron turnover in the Tf pool during the hypoferremic phase was also similar to control rates. Therefore, hypoferremia did not result from an accelerated removal of iron from the plasma Tf pool as previously suggested. These results implicate an impaired return of reticulo-endothelial (RE) system processed iron to the Tf pool during the hypoferremia. / The kinetics of iron processing by the RE system were studied by labeling the RE compartments with ('59)Fe-labeled denatured red blood cells. Uptake and redistribution of the label indicated the RE-processed iron was not returned to the plasma Tf pool during the hypoferremia. Fractionation of hepatic cellular compartments showed that this impaired release of iron resulted from a preferential incorporation of heme-derived iron into the intracellular ferritin pool and this produces the hypoferremia. / The role of ceruloplasmin (ferroxidase I,EC.1.16.3.1) (Cp) in iron metabolism during meningococcal infection was investigated. Plasma Cp ferroxidase activity was found to increase greatly in mice during the convalescence phase. Ferroxidase-deficient mice became hypoferremic as a result of an impaired return of heme-derived iron to plasma Tf. This hypoferremia was associated with an increased resistance to Neisseria meningitidis infection, an effect readily reversed by Cp supplementation or iron addition. This implicates Cp as an important component in the regulation of the plasma Tf iron pool. It also suggests that the increased levels of Cp during the acute phase response may serve to restore normal plasma Tf iron levels.

The temperate bacteriophages Mu and D108 catalyze and regulate the transposition of their 37 kb genomes through several specific protein-DNA interactions. Two early phage encoded gene products directly involved in this process include the transposase (gene A) and ner proteins. / We have cloned, overexpressed and visualized the ner proteins of both phages under the control of the lac UV5 transcriptional promoter and determined (using the band competition assay) that each is a site specific DNA-binding protein. We also physically mapped these ner operator sites by DNase I-footprinting and found minor DNA sequence conservation. Analysis of the DNA sequence of the D108 ner gene revealed little homology at the DNA level but up to 48% conservation of the amino acids of the ner proteins. However, the ner proteins could not specifically bind to each other's operators. Competition experiments and the spatial organization and strength of the early and repressor promoters suggests the evolution of a stronger binding D108 ner protein and hence a difference in the manner that these phages may regulate the expression of their early and repressor gene promoters. In addition, gel filtration experiments showed that both purified proteins behave as monomers in solution with a native molecular weight of 8 kDa. / We have also cloned and visualized proteins containing various amino terminal portions of the D108 transposase under lac UV5 transcriptional control and shown that the 13 kDa amino terminus (which may form two bi-$ alpha$-helical structures) contains a two component sequence specific DNA-binding domain. In addition, we have purified a Mu A$ sp prime$-$ sp prime$bla fusion protein containing 13 kDa of the amino terminus of the Mu transposase and shown that it can bind to the Mu att R site but in a different manner compared to the entire 70 kDa transposase. This binding suggests the possible recognition of the sequence 5$ sp prime$-PuCGAAA-3' which appears to be conserved at the ends of all class II transposons.

This thesis concerns the mechanisms responsible for preferential translation of reovirus mRNA in infected cells. The absence of a poly(A) tract at the $3 sp prime$-end is one of the structural differences that distinguishes reovirus mRNA from cellular mRNA. Addition of free poly(A) inhibits in vitro translation at the level of initiation of protein synthesis, probably by competition between free poly(A) and mRNA. Reovirus mRNA is resistant to this inhibition and this property might confer a translational advantage to the viral mRNA. Another difference between reovirus mRNA and cellular mRNA is the absence of a $5 sp prime$-cap structure on late viral mRNA. This mRNA is translated much more efficiently in lysates from infected than uninfected cells, even in the absence of inhibition of host cell protein synthesis. Addition to uninfected cell lysates of proteins from infected cells stimulates translation of late viral mRNA without effect on other mRNAs; the presence of the sigma 3 viral protein is shown to account for the stimulation of translation. Sigma 3 binds to ribosomes, probably during initiation of protein synthesis, but apparently does not interact directly with mRNA. The sigma 3 protein exists in multiple forms, differing in isoelectric point but possessing all the known properties of sigma 3. These forms possibly result from mutations in the gene encoding sigma 3. The cloned gene which encodes sigma 3 was expressed in L cells without apparent detrimental effect on the cells. Lysates prepared from these cells translate late viral mRNA with an increased efficiency. The sigma 3 protein is associated with the ribosomes in these cells even if no viral mRNA is present. It is suggested that the sigma 3 protein acts as a viral-specific initiation factor of protein synthesis.

An ORF encoding a polypeptide with homology to protein phosphatases 2C (PP2Cs) was identified in the genomes of Salmonella enterica serovar Typhi strains CT18 and Ty2. Analysis of the amino acid sequence revealed an N-terminal segment containing motifs of the catalytic domain of PP2Cs and a C-terminal segment with an unknown function. This protein, termed PrpZ, was expressed as a histidine-tagged fusion protein (PrpZHis) and the purified protein was analyzed for its ability to dephosphorylate various substrates. The Mn2+/Mg2+-dependent phosphatase activity of PrpZHis was inhibited by EDTA and sodium fluoride but unaffected by okadaic acid, indicating that PrpZ is a PP2C. The ability of PrpZHis to remove the phosphoryl group from phosphotyrosine residues was assessed by measuring the release of Pi from phospho-Tyr peptides and MBP. Altogether these data indicate that PrpZ has all the features of a PP2C with dual substrate specificity, in vitro.

Zinc cluster proteins are a major class of transcriptional regulators in Saccharomyces cerevisiae. This protein family includes Uga3 and Dal81, which are both required for the activation of genes involved in the catabolism of gamma-aminobutyrate (GABA). We discovered that the region of Uga3 encompassing amino acids 130 to 300 likely corresponds to the regulatory domain of the protein. We also determined that both the region encompassing amino acids 300 to 350 and the C-terminus of Uga3 are required for activation. Additionally, activation by Uga3 can occur in strains unable to completely degrade GABA or lacking the GABA permease Uga4. Uga3 can form homodimers in vivo, both in the presence and in the absence of GABA. However, it seems that Da181 cannot form heterodimers with Uga3 nor interact with the UGA1 promoter. Furthermore, Dal81 is no longer required for activation if Uga3 is overexpressed or mutated to a constitutively active form.

Recent studies have shown that several DNA-binding motifs are conserved between prokaryotes and eukaryotes, which may indicate the importance of their particular structure/function relationship. An emerging family of highly conserved DNA-binding proteins consists of the following members: the Ner (negative early repressor) proteins of transposable coliphages Mu and D108, of Pseudomonas aeruginosa phage D3112 and of Haemophilus influenzae; the Nlp (Ner-like protein) proteins of Escherichia coli, Neisseria meningitides, Salmonella typhimurium, Photorhabdus luminescence and a number of other prokaryotes; and finally the TMF (TATA-element modulatory factor) proteins of humans, mice, rats and other eukaryotes. / Our laboratory previously established that the E. coli Nlp protein (also known as SfsB, Sugar fermentation stimulation B) was a transcribed nonessential gene in E. coli. I have further established by Northern blot analysis that nlp is expressed as a monocistronic mRNA of approximately 320 nucleotides, and defined the precise nlp/sfsB transcriptional initiation site by primer extension. I also expressed and purified Nlp to homogeneity from a cell-free extract and showed, by electrophoretic mobility assay, that Nlp binds DNA with specificity for the transcriptional regulatory regions of two operons involved in sugar metabolism, mal and lac. E. coli Nlp shares high sequence similarity with the Ner proteins of coliphages Mu and D108 (~62%) and with an 85 amino acid region of the human TMF protein (~30%, named 'TMF'). The high sequence similarity of this human 'TMF' to these regulatory bacteriophage and bacterial proteins suggests the existence of an evolutionarily conserved functional motif. 'TMF' was cloned by PCR from a HeLa cDNA library, over-expressed in E. coli and purified by affinity chromatography. Electrophoretic mobility shift assays suggest specific, although weak, binding of this short 'TMF' protein to the binding site of the larger full length TMF. The Nlp protein acts as a positive activator of several sugar-metabolizing operons (such as mal) in E. coli, but only in strains with cya-crp *1 mutations. I have shown that Mu Ner and 'TMF' are also capable of acting as positive activators of maltose metabolism in the cya -crp*1 mutant, suggesting that the similarity is more than just sequence based. A malPQ::lacZ fusion strain was constructed and beta-galactosidase assays showed that Nlp has a positive effect on the expression of the malP promoter. RNA dot blot analyses further showed that Nlp increases the expression of the malT regulator gene as well as the malF and malK genes (involved in maltose transport). These results give ins

Virus-specific CD8+ T cells have been implicated in the control of acute HIV and SIV infections. Although present in chronic HIV-1 infection, CD8+ T cells exhibit impaired functions due to unidentified molecular signatures. This has urged researchers to revisit parameters that define efficacious CD8+ T cell responses in HIV-1. To this date, polyfunctionality has emerged as the key feature of CD8+ T cell efficacy in chronic infection. Moreover, it is established that T-cell Receptor (TCR) diversity in HIV-specific CD8+ T cells plays a critical role in controlling viremia. Although TCR repertoire studies have been performed in the context of several acute and persistent viral infections including HIV-1, longitudinal studies that aim to study the turnover of the HIV-specific CD8+ TCR repertoire and link HIV-specific CD8+ T cell clonotypes to functional profiles have been limited. We therefore aimed to 1) define a molecular signature of CD8+ T cell exhaustion in HIV-1 infection, 2) study the effect of antigen load and, 3) the effect of viral sequence diversification on the clonality, functionality, and phenotype of HIV-specific CD8+ T cells. Our first set of results identified PD-1 as a molecule of exhaustion on HIV-specific CD8+ T cells and showed a positive correlation with viral load. Interestingly, blocking PD-1 interaction with its ligand alleviated the functional dysfunction of HIV-specific CD8+ T cells. This set of data prompted us to further examine the effect that "antigenic absence" has on the fate of HIV-specific CD8+ T cells. For this, we chose two circumstances; the institution of HAART and emergence of HIV-specific CD8+ T cell epitope escape. Our second set of data provided clear evidence of a HAART-mediated functional reconstitution of the HIV-specific CD8+ T cell compartment on the clonal level. This was attributed by different mechanisms, namely the improvement of function of persisting clonotypes and the recruitment of new clonotypes which were poly / Les cellules T CD8+ sont impliquées dans le contrôle des infections virales aiguës tels que les virus VIS et VIH. Bien que ces cellules T CD8+ spécifiques au VIH persistent durant la phase chronique de l'infection, leur fonctionnalité semble être altérée par un mécanisme non identifié. Cela a exhorté les chercheurs à revoir les paramètres qui définissent l'efficacité réelle des lymphocytes T CD8+ en générale et surtout lors d'infection par le VIH. À date, il est admis que la polyfonctionnalité des cellules T CD8+ soit l'élément clé de l'efficacité de la réponse immunitaire durant l'infection chronique. De plus, il est établi que la diversité du récepteur de cellule T (RCT) occupe un rôle important et crucial dans le contrôle de la virémie. Bien que plusieurs études sur le répertoire des RCT aient été menés par plusieurs groupes dans le contexte des infections aigues et chronique par le VIH, il n'existe néanmoins pas des données combinant la fluctuation et la fonction du répertoire spécifique au VIH au cours de la progression de la maladie. De ce fait, il était important de réaliser des études longitudinales qui ont pour but principal l'étude de la régénération du répertoire T CD8+ spécifiques au VIH durant les différentes phases de l'infection. Dans mes travaux de doctorat, nous avons exploré les mécanismes responsables des l'inefficacité de cellules CD8+ cours de l'infection par le VIH. En utilisant plusieurs techniques immunologiques et génétiques, nos objectifs visaient à : 1) Définir une signature moléculaire d'épuisement, 2) Étudier l'effet de la charge virale et, 3) Caractériser l'effet de la diversification des séquences virales, sur la clonalité, la fonctionnalité, et le phénotype des cellules T CD8+ spécifiques au VIH. Nous avons en premier lieu identifié le rôle de la molécule PD-1, une des molécules régulatrices qui joue un rôle critique dans le contrôle de la réponse immunitai