The true prevalence of hepatitis B virus (HBV) infections in Northern Canada is underreported owing to the lack of occult-HBV studies. Clinical outcomes of HBV infections are variable; such that active disease is rare in Canadian and Greenlandic Inuit, yet hepatocellular carcinoma (HCC) is prevalent among genotype F (HBV/F) infected Alaskan Natives. The purpose of this study is to determine the true extent of occult infections in Northern Canada and the rate, nature and regional susceptibility of HBV genomic mutations among circumpolar indigenous populations. The occurrence of occult infections in 700 archived serum samples from Northern Canada was determined by viral DNA amplification. In addition, HBV mutational analysis was performed on 15 indigenous peoples infected by one of 3 HBV genotypes (B6, D and F), which are associated with varying outcomes, including inactive liver disease and HCC. Phylogenetic analyses and genetic variation was investigated with full-length HBV genomes. The results show 3.8% of 700 study subjects to be occult-HBV positive, with half of them infected with HBV/A. This study also reveals HBV/F strains contain deletions and substitutions associated with adverse outcomes, while HBV/D and B6 sequences lacked these mutations and contained mutational patterns associated with a benign outcome. The genetic diversity within the dominant and subclonal population levels are higher for HBV/B6 strains compared to HBV/D and HBV/F strains. The increased genetic variability found in virus associated with inactive disease may be indicative of an escape mechanism used by HBV to evade the host immune response or simply co-evolution between the virus and its host. These observations may demonstrate the association between differing clinical outcomes and infecting genotype in circumpolar indigenous populations.

Lassa virus (LASV) is the causative agent of a hemorrhagic fever endemic in West Africa, which results in over 200,000 infections and 3000 to 5000 deaths annually. It has previously been demonstrated that a live attenuated recombinant Vesicular Stomatitis Virus expressing the LASV glycoproteins (VSVΔG/LVGPC) is protective in a lethal non-human primate model of Lassa fever. The objective of the present study was to compare the immune response to the VSVΔG/LVGPC vaccine to a novel non-replicating vaccine candidate based on Lassa virus-like particles (VLPs) in the mouse. Additionally, there is a lack of an adequate small animal model for Lassa fever, and LASV is not lethal in immunocompetent mice. We thus investigated the role of the immune system during LASV infection using various immunodeficient mice. In the present study, the VSVΔG/LVGPC vaccine was shown to induce an adaptive immune response, as determined by ELISA and a cytokine ELISPOT assay in immunocompetent mice. The response was stronger with increasing vaccine doses and the number of booster injections. Additionally, the cytokine response preferentially shifted towards epitopes from the N-terminal part of the LASV glycoprotein following the booster injections. A novel vaccine based on Lassa VLPs was also generated, but the immune response to this vaccine candidate was much lower than for the VSVΔG/LVGPC vaccine, even when the VLPs were combined with an adjuvant. Furthermore, we found that mice deficient in the interferon system are susceptible to lethal LASV infection. Complete lethality was observed in STAT1 deficient mice, which are unresponsive to Type I and Type II interferons. Moreover, partial lethality was observed in IFN-[gamma] deficient mice. In contrast, mice lacking major components of the adaptive immune system, as well as wild-type mice, did not show any apparent signs of disease. In conclusion, these results suggest that both the VSVΔG/LVGPC and Lassa VLP vaccine candidates can stimulate an adaptive immune response in the mouse, although the immune response is stronger for the VSVΔG/LVGPC vaccine than for Lassa VLPs. Furthermore, we have established a novel mouse model of LASV infection and lethality, which could potentially be used for vaccine and drug testing against LASV.

The Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tripartite negative-strand RNA virus (NSRV) capable of causing severe hemorrhagic fever within humans. The segmented RNA genome of CCHFV does not exist as naked RNA, but is instead completely encapsidated by a nucleocapsid protein (NP). Very little is known about the molecular mechanism underlying viral NP–RNA encapsidation among the Bunyaviridae. This thesis demonstrates that the CCHFV NP exists as a monomer in solution, an unusual characteristic among NSRVs, even in the absence of RNA. Molecular interactions between recombinant CCHFV NP and ssRNA/dsRNA versions of the short 5’–3’ panhandle formed by the small (S) segment of the CCHFV genome were analyzed by gel electrophoresis mobility shift assays (EMSA) and demonstrated that CCHFV NP failed to bind to the ssRNA version of the short 5’–3’ panhandle structure. This thesis research provides the basis for a broader understanding of the CCHFV NP structure, mechanisms behind RNA encapsidation and the molecular basis of interaction between the short 5’–3’ RNA S-segment panhandle and CCHFV NP.

<p>A fraction of HIV-1 patients are able to successfully control the virus and avoid developing AIDS. It has become increasingly clear that variations in the immune response during the initial days of acute infection including the period of peak viral replication determine long term differences in disease outcomes. While the precise factor(s) necessary and sufficient for protection from AIDS is as yet unidentified, a number of factors have been correlated with protection from AIDS. Among these are the presence of a strong proliferative and multifunctional T-cell response as well as the HLA allele status of a patient. Therefore the goal of this thesis project was to 1) broadly identify the major contributors to the proliferative and multifunctional T-cell response during acute infection with HIV, 2) examine the durability of these responses and 3) elucidate the gene regulation pathway(s) by which HLA allele status determines disease outcomes.</p><p>In order to identify the major contributors to the proliferative and multifunctional T-cell response to HIV we utilized PBMC samples from a cohort of acutely infected HIV patients in the Duke and University of North Carolina infectious disease clinics. These samples were stimulated in vitro with peptides representing the HIV clade B consensus sequence and the T-cells were analyzed for proliferation and multifunctionality. Through this analysis we identified CD4+CD8+ (DP) T-cells as overrepresented within the proliferative response and the primary contributor to multifunctionality. Additionally, the acute multifunctional T-cell response was highly focused on the Nef, Rev, Tat, VPR and VPU sections of the HIV proteome. We also discovered similar response patterns among a cohort of HIV controllers recruited from the Duke infectious disease clinic. In fact, the frequency of multifunctional DP T-cells was inversely correlated with viral loads among the controller cohort.</p><p>Having identified DP T-cells as HIV responding cells of interest, we next examined their durability following the removal of widespread antigenic stimulation via administration of HAART. Utilizing longitudinal samples from the acute HIV cohort we again examined T-cell proliferation and multifunctionality at approximately 24 weeks and 104 weeks post infection among patients. This experiment demonstrated that among patients who initiated HAART during acute infection there was a significant reduction in the frequency of multifunctional DP T-cells at 24 and 104 weeks post infection compared to study entry. Meanwhile the proliferative DP T-cell response was maintained longitudinally. Additionally, these patients did not exhibit the previously described increase in frequency of multifunctional CD8 T-cells as infection progressed to the chronic phase. Although the majority of patients initiated HAART during the acute stage of infection, a minority delayed HAART initiation for various lengths up to and including study cessation. Among this group of patients the frequency of multifunctional DP T-cells was maintained longitudinally. Therefore, the early initiation of HAART reduces long term frequencies of multifunctional DP T-cells while delayed HAART initiation leads to a durable multifunctional DP T-cell response. Since HIV controllers with higher frequencies of multifunctional DP T-cells maintain lower viral loads, early HAART initiation may be detrimental to the development of immune cells capable of controlling the virus.</p><p>Finally, we examined the effect HLA alleles have on gene regulation during the initial interactions between HIV and the host immune system. This work employed 2 HIV negative patient cohorts. One cohort expressed HLA-B*35 which has previously been shown to correlate with rapid progression to AIDS following infection with HIV. The second cohort expressed HLA-B*57 which has been associated with long term non-progression following infection with HIV. PBMCs from each group were infected with HIV in vitro. Twenty-four hours after infection these cells were sorted into CD4+ T-cells, CD8+ T-cells and NK-cells. Following cell sorting, mRNA was isolated and interrogated for expression changes using whole genome microarrays. This analysis revealed HLA allele specific differences in the magnitude by which CD4+ T-cells, CD8+ T-cells and NK-cells activate the interferon response pathway following exposure to HIV.</p><p>In total, these findings provide insight into the cell types responsible for significant portions of the acute immune response to HIV and the mechanisms by which individuals protected from progression to AIDS differ from their peers.</p> / Dissertation

The interferon-induced protein kinase RNA-activated (PKR) is activated after virus infection but not during human immunodeficiency virus type-1 (HIV-1) infection of lymphocytes. The TAR RNA binding protein (TRBP) counteracts the inhibitory effects of PKR on HIV-1 expression and replication. In U251MG astrocytes, which are low TRBP producers, PKR is activated upon HIV-1 transfection and viral production is low. Astrocytes overexpressing TRBP express more viral proteins. Expression of adenosine deaminase acting on RNA-1 (ADAR1)-p150 and its binding to PKR are increased during high HIV-1 replication. ADAR1 also reverses PKR inhibition of HIV-1 expression and production in HEK 293T and astrocytes. DNA- and RNA-binding domains of ADAR1 are required for PKR inhibition. Surprisingly, the PKR activator protein (PACT) also reversed PKR inhibition of HIV-1 expression in HEK 293T. These results indicate that three double stranded RNA binding proteins, TRBP, ADAR1 and PACT belong to a multiprotein complex that inhibits PKR function during HIV-1 infection. / La protéine kinase induite par l'interféron activée par l'ARN (PKR) est activée après une infection virale, mais pas durant l'infection des lymphocytes par le Virus de l'Immunodéficience Humaine de type-1 (VIH-1). La protéine de liaison à l'ARN TAR (TRBP) empêche l'effet inhibiteur de PKR sur l'expression et la réplication du VIH-1. Dans les astrocytes U251MG, exprimant faiblement TRBP, PKR est activée par le VIH-1 et la production virale reste basse. Des astrocytes qui surexpriment TRBP produisent plus de protéines virales. L'expression de l'adénosine déaminase agissant sur l'ARN-1 (ADAR1)-p150 et sa liaison à PKR augmentent quand le VIH-1 se réplique activement. ADAR1 inverse l'inhibition de l'expression et de la production du VIH-1 par PKR. L'inhibition de PKR nécessite les domaines de liaison à l'ARN et à l'ADN d'ADAR1. Étonnamment, l'activateur de PKR (PACT) inverse aussi l'inhibition du VIH-1 par PKR dans les HEK 293T. Ces résultats indiquent que trois protéines liant l'ARN double brin, TRBP, ADAR1 et PACT forment un complexe multiprotéique qui inhibe la fonction de PKR pendant l'infection du VIH-1.

Dendritic cells (DC) have a central role in cell-mediated immunity and they are today in the middle of many immunotherapy strategies. Recently, a clinical trial was initiated at the Montreal Chest Institute, to evaluate the effect of an immunotherapy (AGS-004) containing autologous DC to amplify the T cell immune responses of HIV-1-infected subjects. However, concerns have been raised that B cell activation following DC immunotherapy may lead to the development of autoimmune diseases. Here, we studied the safety, patient tolerance and changes in total B cells and B cell subsets following administration of AGS-004 in ten HIV-1 subjects receiving antiretroviral therapy (ART). Clinically, AGS-004 was safe, well tolerated and caused few mild side effects. Moreover, CD4 and CD8 cell counts and HIV-1 viral load were unchanged throughout the 48-weeks follow-up study period. In addition, total B cells and B cell subsets, which were measured as an indicator of the immune activation status, did not change over time, except that the proportion of B memory cells significantly increased after receiving the AGS-004 immunotherapy (P=0.005). Collectively, these data show that the AGS-004 is relatively well tolerable and induces an increase in B memory cells. Further investigations would need to be done to confirm the presence of an activated immune status including functional properties of these B memory cells and antibody measurements. / Les cellules dendritiques (DC) ont un rôle central dans l'immunité à médiation cellulaire et elles sont aujourd'hui au centre de nombreuses stratégies d'immunothérapie. Récemment, un essai clinique à base des DC autologues, a été initié à l'Institut Thoracique de Montréal pour évaluer l'effet d'une immunothérapie (AGS-004) à amplifier les réponses immunitaires des lymphocytes T chez les sujets infectés par le VIH-1. Cependant, le risque potentiel de l'activation des lymphocytes B, menant au développement de maladies auto-immunes, a été soulevé suivant des immunothérapies à base de DC. Dans le présent travail, nous avons évalué l'innocuité, la tolérance et les changements des lymphocytes B et leurs sous-populations après DC immunothérapie chez dix sujets infectés par le VIH-1 et recevant une thérapie antirétrovirale. Cliniquement, l'AGS-004 était sans danger, bien tolérée avec quelques effets secondaires bénins. De plus, les valeurs de la charge virale et les décomptes lymphocytaires CD4 et CD8 n'ont pas été modifiées tout au long de la période de suivi de 48 semaines. En outre, les pourcentages de lymphocytes B totaux et leurs sous populations n'ont pas changé, à l'exception des lymphocytes B mémoires qui ont considérablement augmenté après l'immunothérapie (P=0.005). Collectivement, ces résultats montrent que l'AGS-004 est relativement bien tolérée et induit une augmentation des lymphocytes B mémoires. Il demeure donc intéressant de confirmer ces résultats et d'étudier la fonction de ces lymphocytes de manière plus poussée ainsi que de mesurer la production des anticorps. fr

The reverse transcriptase (RT) enzyme of human immunodeficiency virus 1 (HIV-1) is an RNA- and DNA-dependent DNA polymerase that also exhibits RNase H activity. The polymerase and RNase H active sites of RT are structurally linked through the connection domain. RT is a major antiretroviral target but the development of viral resistance often leads to therapy failure. Genotyping protocols detect mutations in the polymerase active site that confer resistance to nucleoside analogue RT inhibitors such as zidovudine (AZT). It has been shown however, that mutations outside of this region, such as the N348I substitution in the connection domain, also confer resistance to AZT. Here, we have investigated the mechanism of N348I drug resistance. Our biochemical results have shown that the N348I mutation affects drug susceptibility by reducing nucleic acid substrate binding in the RNase H domain of RT. The N348I mutation also compensates for enzymatic deficits introduced by other resistance-conferring mutations, thus providing a rationale for its selection pattern in vivo.Additionally, we have investigated the mechanism through which INDOPY-1, a newly discovered antiretroviral agent, disrupts RT function. Although INDOPY-1 is not a nucleoside analogue, it can compete with incoming nucleotides and select for mutations at the site of nucleotide binding. Based on these observations, we propose that this antiretroviral agent belongs to a new class of RT inhibitors that we name "Nucleotide-competing RT Inhibitors". Finally, cellular ATP was shown to enhance the potency of INDOPY-1, a property never before seen with any other RT inhibitors. Based on the biochemical properties of this interaction, we propose to describe ATP as an "enhancer" of INDOPY-1-mediated RT inhibition. ATP enhances the binding of INDOPY-1 to RT by "capping" the inhibitor in the polymerase active site.Work presented in this thesis characterizes a novel mechanism of resistance to clinically approved RT inhibitors. It also describes, for the first time, a new class of RT inhibitors that function through a unique mechanism of action. / La transcriptase inverse (RT) du VIH-1 est une polymérase d'ADN et d'ARN qui est aussi capable de dégrader l'ARN viral durant la polymérisation. Le domaine de connextion de la RT crée un lien entre le site actif de la polymérase et le site actif d' RNase H de cette enzyme. La RT est une cible majeure de la thérapie contre le SIDA. Mais, à cause du taux de mutation élevé du virus, le développement de résistance aux médicaments est rapide, ce qui engendre la neutralisation des effets avantageux des thérapies antivirales. Les analogues de nucléosides, comme zidovudine (AZT), sont souvent utilisés durant la thérapie anti-SIDA. Les tests génotypiques qui détectent la résistance virale incluent seulement le domaine de la polymérase de la RT. Par contre, il a été démontré que des mutations dans les domaines de la connextion, par example le mutation N348I, peuvent causer la résistance à AZT. Nous avons étudié le mécanisme de résistance à la mutation N348I. Nos resultats biochimiques montrent que N348I a un effet négatif sur l'affinité de l'ARN pour le site actif d'RNase H. Nous montrons aussi que la mutation N348I peut compenser pour des domages causés par d'autres mutations. Ceci peut expliquer l'apparence de cette mutation in vivo. Nous avons aussi étudié le mécanisme d'action d'INDOPY-1, un nouveau agent inhibatoire de la RT. Malgré que l'INDOPY-1 n'est pas un analogue de nucléoside, cet agent peut entrer en compétition avec les nucléotides et les mutations de résistance sont selectionnées dans le site d'association des nucléotides. C'est ainsi que nous avons proposé que l'INDOPY-1 appartient à une nouvelle catégorie d'inhibiteurs que nous nommons «Nucleotide-competing RT inhibitors». Un autre trait caractéristique de l' INDOPY-1 est que sa capacité d'inhibition peut être augmentée par l'ATP cellulaire. Basé sur ceci, nous décrivons l'ATP comme étant le «renforceur» de l'INDOPY-1. Ce mécanisme de renforcement se produit lorsque l'ATP «encapsule» l'INDOPY-1 dans le site actif de la polymérase de la RT.En conclusion, nous avons characterisé un nouveau mécanisme de résistance aux analogues de nucléosides. Nous avons aussi découvert le premier composé d'une nouvelle classe d'agents inhibatoires de la RT.

Over the last 30 years, the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) mêlée has endured despite valiant efforts made by the research community. Although great strides in treatment efficacy have been made, an effective vaccine remains beyond our reach. Anti- HIV microbicides, (topically applied agents to prevent the transmission of HIV), offer an infection prevention strategy that may buy us time until an effective vaccine is discovered. Indeed, antiretroviral (ARV)-based microbicides have proven more effective than any vaccine clinical trial to date. Improved formulations, including combination ARV- based microbicides, may prove to be even more clinically efficacious in the future. However, the pre-clinical evaluation of candidate microbicide ARVs is prudent, lest a formulation proves inimitably toxic or useless during human trials. Of course, pre-clinical drug assessments have limitations; what we learn from in vitro tissue culture experiments does not necessarily translate seamlessly to in vivo efficacy. Pre-clinical assessment may, nevertheless, aid in advancing the best possible candidate ARVs, permitting each candidate microbicide ARV to be ranked in the context of better toxicity, efficacy, and drug resistance development profiles. The following thesis describes the pre-clinical assessment of the lead candidate microbicide ARVs, tenofovir (TFV) (a nucleotide reverse transcriptase inhibitor (NtRTI)), dapivirine (DAP) (a nonnucleoside reverse transcriptase inhibitor(NNRTI)), and BMS- 599793, an HIV-1 entry inhibitor. How these drugs perform as HIV-1 infection preventatives in the context of wild type (WT) and drug resistant (DR)HIV-1 transmission in vitro is focused upon. / Au cours des 30 dernières années, le virus de l'immunodéficience humaine (VIH), agent causative du syndrome d'immunodéficience acquise (SIDA) a enduré malgré les vaillants efforts déployés par la communauté de la recherche. Bien que de grands progrès au niveau de l'efficacité du traitement ont été accomplis, un vaccin efficace reste hors de notre portée. Les microbicides anti-VIH (agent topiques appliqués pour prévenir la transmission du VIH) offrent une stratégie de prévention des infections et peuvent probablement nous acheter du temps jusqu'au moment ou un vaccin efficace sera découvert. En effet, les antirétroviraux (ARV) lorsqu'utilisés comme microbicides sesont révélés plus efficaces que tout essai clinique du vaccin, à ce jour. Des formulations améliorées, y compris les combinaisons d'ARV à base de microbicides, pourront s'avérer encore plus efficaces dans l'avenir. Cependant, l'évaluation préclinique des candidats ARV de microbicide est une précaution importante lorsque nous prenons en considération le fait que certains ARV peuvent être toxiques ou inutiles au cours d'essais sur les humains. Bien entendu, les évaluations précliniques de drogues ont des limitations; ce que nous apprenons à partir d'expériences de culture de tissus n'est pas nécessairement traduit parfaitement in vivo au point de vue de l'efficacité. L'évaluation préclinique peut, néanmoins aider à faire avancer le candidat ARV le meilleur, en permettant à chaque candidat microbicide d'être classé dans le contexte de la toxicité de l'efficacité et des profils de résistance. La thèse qui suit décrit l'évaluation préclinique de la ténofovir (TFV) (un nucléotide inhibiteur de la transcriptase inverse (NtRTI), de la dapivirine (DAP) (un inhibiteur non-nucléosidique de la transcriptase inverse (INNTI)) et de la BMS-599793, un inhibiteur d'entrée du VIH-1. Comment exercer ces médicaments préventifs d'infection au VIH-1 dans le contexte de virus de type sauvage (WT) et virus résistant aux médicaments est le thème de la présente thèse.

Micronutrients are increasingly appreciated as potent immunomodulators. Long used to 'treat' measles virus (MeV), vitamin A was recently shown to act through RIG-I to up-regulate type 1 interferons. Similar actions are seen with some (mumps virus and CDV) but not all members of the Paramyxoviridae. However, children infected with respiratory syncytial virus (RSV), a close relative of MeV, do worse when given pharmacological doses of vitamin A. RSV is known to elicit a pathologic Th2-biased response. Vitamin A also has a strong Th2-deviating influence. Our primary objective was to develop a small animal model of vitamin A deficiency and sufficiency in which we could assess the impact of vitamin A status and supplementation on RSV infection in vivo. Such a model will be of great use to characterize the mechanism of action of retinoids in this infection. We succeeded in the development of this model by restricting dietary retinol through 2 generations of BALB/c mice (ie: deficiency state) and introducing novel means to reliably attain a state of consistent vitamin A supplementation (ie: reconstituted &amp; excess states). Preliminary data using this model suggested that there were marked differences in RSV pathology between deficient and sufficient mouse groups. Like the apparent situation in humans, infection in the vitamin A deficient mice was paradoxically less severe than in mice with a positive vitamin A status. This model and the data generated with it may be of particular interest in regions with diets high in vitamin A (North America in particular). Historically, very little attention has been given to possible negative effects of micronutrient 'over-nutrition'. The limited human data and the preliminary data from our new model brings into question whether or not we are 'priming' ourselves for more severe RSV infection than would otherwise occur. The data generated in this model may also be highly relevant to guide supplementation efforts in regions of the world that currently have less access to vitamin A. / Les micronutriments sont des plus appréciés comme immunomodulateurs puissants. Longtemps utilisé pour traiter measles virus (MeV), la vitamine A a été récemment montré à agir par le biais de RIG-I pour réguler les interférons de type 1. Des actions similaires sont observés avec certains (canine distemper virus, CDV), mais pas tous les membres du Paramyxoviridae. Cependant, les enfants infectés par des voies respiratoire, respiratory syncycial virus (RSV), un proche cousine de MeV, faire pire lorsqu'il est administré des doses pharmacologiques de vitamine A. Le RSV est connu pour induire une résponse pathologique biaisée au Th2. La vitamine A est également une forte influence pour la Th2 déviation. Notre principal objectif était de développer un modèle animal de la carence en vitamine A et la suffisance dans lesquels nous pourrions évaluer l'impact du statut en vitamine A et de la supplémentation sur l'infection à RSV in vivo. Un tel modèle sera d'une grande utilité pour caractériser le mécanisme d'action des rétinoïdes dans cette infection. Nous avons réussi dans le développement de ce modèle en limitant le rétinol alimentaires grâce à deux générations de souris BALB / c (ie: état de carence) et en introduisant de nouveaux moyens de atteindre un état fiable de vitamine A cohérente supplémentation (ie: les Etats reconstitué &amp; excès). Les données préliminaires utilisant ce modèle suggéré qu'il y avait des différences marquées dans la pathologie du RSV entre les groupes de souris déficientes et suffisante. Comme la situation apparente dans les humains, l'infection dans la vitamine A des souris déficientes en était paradoxalement moins sévère que dans les souris avec une vitamine positifs d'un statut. Ce modèle et les données générées avec elle peut être d'un intérêt particulier dans les régions où les régimes son riches en vitamine A (Amérique du Nord en particulier). Historiquement, très peu d'attention a été accordée aux effets négatifs possibles des micronutriments sur-nutrition. Les données humaines limitées et les données préliminaires de notre nouveau modèle remet en question si oui ou non nous sommes d'amorçage nous-mêmes pour l'infection à VRS plus sévères que se produirait autrement. Les données générées dans ce modèle peut également être très pertinentes pour guider les efforts de la supplémentation dans les régions du monde qui ont actuellement un accès moindre à la vitamine A.

The Hepatitis C Virus (HCV) is a major public health concern considering it infects around 3% of the global population and there is currently no virus-specific therapy or vaccine. Several virally-encoded proteins have been identified as potential targets for inhibitor design including the helicase-containing nonstructural protein three (NS3). / Helicases are enzymes that are responsible for the separation of double-stranded nucleic acids prior to most processes of nucleic acid metabolism. It is well documented that disabling the HCV helicase through mutation results in the arrest of the viral lifecycle. The aim of our work was to characterize the details of the molecular biology of the HCV helicase. / We have cloned the helicase domain of NS3 and have conducted studies on its NTPase, DNA binding, and unwinding activities using a collection of biochemical assays. We were able to describe the previously unknown interface that exists between the helicase and a DNA substrate and identify a basic amino acids cluster (K360-K380) that is important for DNA binding and unwinding. We have shown that the previously uncharacterized Motif IV is responsible for binding at the junction between substrate and product DNA. Furthermore, we have implicated residues within Subdomain 3 (K583-K589) that are likely involved in the strand separation process. / Helicases catalyze the separation of nucleic acid strands using the energy that is derived from the hydrolysis of nucleotide triphosphates (NTPs). This chemical reaction powers conformational changes within the protein, resulting in translocation of the helicase along DNA and RNA substrates to eliminate structural features that shield the genetic code. During the second phase of our study, we used a high-resolution protein footprinting assay, among other biochemical tests, to study the link between NTP hydrolysis and the strand separation process. We found that the dynamic movement of Subdomain 2, which contains both Motif IV (in contact with the DNA) and Motif VI (in contact with the NTP) is responsible for transducing NTP hydrolysis energy into the DNA unwinding activity of HCV NS3. / Tenant compte du fait que le virus de l'hépatite C (HCV) infecte approximativement 3% de la population mondiale et qu'il n'existe ni médicament spécifique ni vaccin pour ce virus, celui-ci représente une inquiétude majeure en santé publique. Plusieurs protéines virales ont été identifiées comme sources possibles pour la création d'agents inhibiteurs, incluant la protéine non-structurale 3 (NS3) qui comprend l'activité de l'hélicase. / Les hélicases sont les enzymes responsables de la séparation des deux brins des acides nucléiques qui précède la plupart des processus métaboliques de ceux-ci. La désactivation par mutation de l'hélicase du HCV provoquant l'arrêt du cycle viral est bien documentée. Le but de notre travail était de caractériser les détails de la biologie moléculaire de l'hélicase du HCV. / Nous avons cloné le domaine de l'hélicase de la NS3. Utilisant une multitude d'analyses biochimiques, nous avons étudié son activité NTPase, ainsi que ses activités de fixation et de déroulement d'ADN. Nous avons pu décrire l'interface jusqu'ici inconnue qui existe entre l'hélicase et un substrat d'ADN, et identifier une grappe d'aminoacides (K360-K380) impliqués dans la fixation et le déroulement d'ADN. Nous avons démontré que le Motif IV est responsable de la fixation à la jonction entre le substrat et le produit d'ADN. Nous avons également identifié des résidus du Sous-domaine 3 (K583-K589) qui semblent très probablement impliqués dans le processus de séparation des brins. / L'hélicase catalyse la séparation des brins des acides nucléiques en utilisant l'énergie dérivée de l'hydrolyse de nucléotides triphosphates (NTP). Cette réaction chimique fournit l'énergie nécessaire aux changements conformationnels de la protéine, responsables du mouvement de l'hélicase le long des substrats d'ADN et d'ARN qui élimine les structures masquant le code génétique. Dans la deuxième étape de notre recherche, nous avons utilisé, parmi d'autres tests biochimiques, une méthode d'analyse d'emprunte protéique à haute résolution pour étudier la relation entre l'hydrolyse des NTPs et la séparation des brins des acides nucléiques. Nous avons découvert que le mouvement dynamique du Sous-domaine 2, qui contient à la fois Motif IV (en contact avec l'ADN) et Motif VI (en contact avec le NTP), est responsable de la transduction de l'énergie produite par l'hydrolyse des NTPs en l'énergie nécessaire pour le déroulement d'ADN du HCV NS3.