The human immunodeficiency virus is presently one of the most significant global health issues to date, with a disease burden that encumbers developing and developed nations alike. Although current antiretroviral therapy can help patients maintain undetectable levels of the virus throughout their bodies, once the treatment is ceased, the virus will rebound and disease progression continues. Thus, modalities to; 1- stop HIV transmission and spread, or 2- eradicate the virus once it is acquired are both urgently needed. In this project, we seek to evaluate and understand the impact of a candidate vaccine therapy that targets the HIV protease cleavage sites (PCS) on viral fitness. Vaccination with this modality in a monkey model induces mutations at virus regions that are intolerant to change, presumably affecting the "fitness" of viral strains recovered from vaccines. Preliminary results of the study show that in the vaccine group (n=11), a disruption to one or more of the HIV protease cleavage sites results in improved maintenance of CD4+ T cells compared to unvaccinated controls (n=5). Furthermore, a correlation between the percentage of PCS mutations and reductions in viral load were seen. Our data indicate that the most common sites of mutation occur at two cleavage regions PCS2 and PCS12. We used site directed mutagenesis to introduce multiple PCS mutations into infectious clones of SIV. Our ongoing studies are evaluating the viral fitness of the SIV mutants in a cell lines and PBMC using competitive viral fitness assays. The data from these studies will help inform in the areas of vaccine and therapy development for HIV-1.

Dans ce travail de thèse, nous avons développé et optimisé une stratégie originale pour la caractérisation des extrémités N-terminales des protéines et des sites de clivages protéolytiques. Elle s’appuie sur la dérivation chimique spécifique des amines N-terminales et nous l’avons adapté à différents types d’échantillons biologiques. L’application de cette stratégie dans des études en biologie nous a permis d’apporter plusieurs éléments de réponse à différentes problématiques. Nous avons ainsi caractérisé les peptides de transit des protéines mitochondriales humaines et ainsi validé/corrigé expérimentalement leurs prédictions dans les banques de données. Nous avons aussi appliqué cette stratégie à l’étude du protéome du parasite P. falciparum. La mise au point de la dérivation N-terminale de protéines immobilisées dans un gel SDS PAGE nous a permis d’étudier le mécanisme d’export des protéines de ce parasite vers sa cellule hôte et de déterminer le rôle des acides aminés impliqués dans cet export. Un réactif de dérivation marqué aux isotopes stables permet d’effectuer des études différentielles des processus protéolytiques que subissent les protéines. Cette stratégie quantitative a été appliquée à l’étude du protéome hépatique du rat soumis au jeûne expérimental. D’autres applications de l’analyse protéomique en biologie sont aussi présentées dans ce manuscrit. / In this manuscript, we describe the development and the optimization of an original strategy for the characterization of protein N-termini and protease cleavage sites. The strategy is based on specific chemical derivation of alpha-amines. We applied this method to the characterization of mitochondrial proteins’ transit peptides which allowed us to experimentally validate/correct their prediction in protein databases. In another study, the strategy was applied to the analysis of the proteome of the malaria parasite Plasmodium falciparum. The optimization of ingel N-terminal derivation and its application to the study of parasite exported proteins allowed us to determine the role of implicated amino acid residues in the signaling and export mechanism of these proteins to the host cell. To enable differential studies of proteolysis, we introduced an isotope labeled derivation reagent. This quantitative method was applied in the context of a study of the rat liver proteome after experimental long-term fasting. Other applications of proteomics in biology are also presented in this manuscript.