Masters of Science / The rapidly expanding field of nanotechnology has been the focus of many biologists with regard to drug delivery. The ability of nanoparticles to enter cellular compartments makes it possible to explore specific treatment strategies for life-threatening diseases such as AIDS. Since HIV primarily infects CD4+ cells, we aim to use CD4 as a selectable marker to deliver pro-apoptotic nano-devices to HIV infected cells. The objective is to selectively induce cell death or apoptosis in CD4+ HIV infected cells. Apoptosis is activated through a number of biochemical pathways. The apoptosis promoting protease, caspase-3 is central to the induction of apoptosis. Caspase-3 is produced as an inactive zymogen and is activated by other proteases through proteolytic cleavage. We take advantage of the fact that HIV-infected cells produce HIV-1 protease, which is responsible for the production of infectious virions through proteolytic cleavage of the HIV proteins, Gag and Pol. Our strategy was to generate a mutant form of the caspase-3 protease that is only cleavable by HIV-1 protease.

Follicular dendritic cells (FDCs) reside in the lymphoid follicles of the secondary lymphoid tissues (sLTs). Following the infection of an individual with human immunodeficiency virus type 1 (HIV-1), viral particles are trapped in massive quantities on the surfaces of FDCs. HIV-1 viral compartments are cell types or tissues between which there is a restriction of virus flow. Compartmentalization of HIV-1 creates numerous sites within the body in which the virus can undergo independent evolution, giving rise to a more diverse total viral population. Given the sessile nature of the FDC, I hypothesized that contrary to common assumptions, FDC-trapped HIV-1 is compartmentalized between different sLTs. Furthermore, given that FDC-trapped HIV-1 represents the major source of virus in the host, I postulated that this compartmentalization would likely impact the diversity of HIV-1 associated with the sLTs. I isolated FDCs, macrophages, and T cells from various sLTs, and sequenced cloned HIV-1 associated with these three cell populations. I subjected the resulting DNA and cDNA sequence data to phylogenetic and other statistical analyses. In support of my hypothesis, I demonstrate that both HIV-1 gp120 and pol sequences cloned from FDCs are compartmentalized between different sLTs. This compartmentalization is even apparent between lymph nodes taken from the same lymph node chain. One of the apparent effects of this compartmentalization is to significantly increase the viral genetic diversity in multiple sLTs when compared with diversity in a single sLT. It also appears that the selective pressures on HIV-1 differ among the sLTs. In addition, when proviruses isolated from macrophages from different sLTs were compared, it was also evident that there is compartmentalization of HIV-1 associated with this cell type as well. Finally, I demonstrate that HIV-1 isolated from an unfractionated population of cells from a single sLT, may be an inadequate representation of the total viral population in that sLT. Taken together, my data suggest that the nature of HIV-1 in the sLTs may be more complex than currently appreciated.

Despite our growing knowledge of virus biology they continue to present a problem to global public health. This problem arises from their high mutation rates that allow them to evade antiviral therapies that we have developed to date. An alternative solution for developing antiviral therapies could be to target host cell factors that are hijacked by the virus. The basis of this hypothesis is that if we can stop the virus from using host cell machinery or from evading host immune mechanisms we could treat the infection more efficiently. With the major research focus being on viral proteins and how we can prevent their functions, there is a lot of work to be done in finding host factors that could be the key to treating an infection. The three themes presented in this thesis broadly focus on this goal. The first theme looks at miRNAs, their interacting partners, and their dysregulation during HCV infection. A microRNA is identified from a small molecule screen of miRNAs that are dysregulated during HCV infection and its role in liver immunometabolism is examined to determine its antiviral potential and identify host factors that could be of interest to target with antiviral therapeutics. The second theme examines the potential of activity-based protein profiling techniques for complementing existing antiviral therapies. An azauracil probe is characterized to examine its ability to interact with viral polymerases and its suitability as a building block for antiviral research or therapies. The final theme uses activity-based protein profiling techniques to study a novel carbamate-hydrazone chemotype and establish its suitability as a chemical probe. The hydrazone probe’s reactivity with the mammalian proteome was determined and its interacting partners were identified using chemoproteomic techniques with an overall goal of examining its suitability for antiviral research. Overall, this thesis uses chemical and molecular biology techniques to present three differing perspectives on how to approach the discovery of host factors and develop novel antiviral therapies.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25515号 / 生博第531号 / 新制||生||70(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 荒木 崇, 教授 野田 岳志, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations. / AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1 (MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies onder andere die induksie van apoptose, die induksie van selsiklus G2 staking, modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr proteine te ondersoek Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie, is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die modulering van geen uitdrukking in die induksie van apoptose is deur TLDA ondersoek. Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en 4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S. Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer, maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief veroorsaak is deur die intrinsieke roete. Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-κB, facilitated nuclear translocation of NF-κB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-κB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-κB translocation, which are the characteristics of pulmonary endothelial cell activation.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage. / The human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immune deficiency syndrome (AIDS). HIV-1 develops a resistance towards the inhibitors used to treat infected patients. It is thus important to identify new targets for the development of novel antiretroviral agents. The aim of our work was to better characterize the programmed -1 ribosomal frameshift which generates the precursor of HIV-1 enzymes. The frameshift occurs at a specific sequence of HIV-1 full-length messenger RNA (mRNA), the slippery sequence, and is performed by a minority of the ribosomes translating this mRNA. The frameshift efficiency is controlled by the frameshift stimulatory signal (FSS), an irregular stem-loop located downstream of the slippery sequence. FSS structure is unfolded by every ribosome translating this region and can refold afterwards. We showed that HIV-1 frameshift efficiency is affected by changes in the rate of translation initiation. We transfected Jurkat-T and HEK 293T cells with a bicistronic reporter that contains the frameshift region of HIV-1 between the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) genes. Rluc is produced by all ribosomes translating this reporter whereas only ribosomes that make a –1 frameshift produce Fluc. The translation of the reporter is initiated via a cap-dependant mode, like the majority of cellular mRNAs. We first determined the effect of three inhibitors of translation initiation. We showed that their presence increases the frameshift efficiency. We next determined the impact of the TAR stem loop, which is located at the 5’end of every HIV-1 mRNA. TAR is known to impair the binding of the small subunit of the ribosome (40S) to the mRNA. TAR also modulates the activity of the double-stranded RNA-dependent protein kinase (PKR). When PKR is activated, it phosphorylates the eukaryotic initiation factor 2 (eIF2), inhibiting translation initiation. The inhibition of PKR has the opposite effect. We studied the effect of TAR on PKR by positioning TAR at a distance of the 5’ end where it cannot interfere with the binding of the 40S. Our results showed that a small amount of TAR, which activates PKR, increases the frameshift efficiency whereas a large amount of TAR, which inhibits PKR, decreases it. A model is presented where the variations of translation initiation modulate HIV-1 frameshift efficiency by altering the distance between the elongating ribosomes. This influences the probability that these ribosomes encounter or not a folded FSS. We next observed the effect of the 5’ untranslated region (UTR) of HIV-1 full length mRNA on its frameshift efficiency. This 5’UTR contains several structured parts, including TAR at the 5’end, which can inhibit translation initiation. This mRNA has a cap and an internal ribosome entry site (IRES) and could then use a cap dependent and an IRES-dependent mode of translation initiation. We replaced the 5’UTR of our bicistronic reporter mRNA by the complete 5’UTR of HIV-1 full-length mRNA or a part of it. Our results showed that the presence of the complete 5’UTR inhibits cap-dependent initiation of translation and increases the frameshift efficiency. Those effects are mostly due to the presence of TAR followed by a Poly(A) stem-loop. We also constructed a tricistronic reporter where the ribosomes translating the luciferases have to use an IRES-dependent initiation mode. The rate of this initiation was low and the frameshift efficiency obtained was also low. We proposed a hypothesis accounting for this situation. We also observed that when both initiation modes are available, the cap-dependent mode seems to be highly favored. Finally, we studied the impact of the Tat viral protein on translation initiation and frameshift efficiency. We showed that the presence of Tat increases translation initiation and decreases the frameshift efficiency. Those effects are more important when TAR is present at the 5’end of mRNA. We propose a model explaining the effects of Tat on translation initiation by the inhibition of PKR and by changes in the expression of cellular proteins that are able to unfold TAR. Our results allow us to better understand the mechanisms controlling HIV-1 frameshift, which will help in the development of drugs targeting the HIV-1 frameshift.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage. / The human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immune deficiency syndrome (AIDS). HIV-1 develops a resistance towards the inhibitors used to treat infected patients. It is thus important to identify new targets for the development of novel antiretroviral agents. The aim of our work was to better characterize the programmed -1 ribosomal frameshift which generates the precursor of HIV-1 enzymes. The frameshift occurs at a specific sequence of HIV-1 full-length messenger RNA (mRNA), the slippery sequence, and is performed by a minority of the ribosomes translating this mRNA. The frameshift efficiency is controlled by the frameshift stimulatory signal (FSS), an irregular stem-loop located downstream of the slippery sequence. FSS structure is unfolded by every ribosome translating this region and can refold afterwards. We showed that HIV-1 frameshift efficiency is affected by changes in the rate of translation initiation. We transfected Jurkat-T and HEK 293T cells with a bicistronic reporter that contains the frameshift region of HIV-1 between the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) genes. Rluc is produced by all ribosomes translating this reporter whereas only ribosomes that make a –1 frameshift produce Fluc. The translation of the reporter is initiated via a cap-dependant mode, like the majority of cellular mRNAs. We first determined the effect of three inhibitors of translation initiation. We showed that their presence increases the frameshift efficiency. We next determined the impact of the TAR stem loop, which is located at the 5’end of every HIV-1 mRNA. TAR is known to impair the binding of the small subunit of the ribosome (40S) to the mRNA. TAR also modulates the activity of the double-stranded RNA-dependent protein kinase (PKR). When PKR is activated, it phosphorylates the eukaryotic initiation factor 2 (eIF2), inhibiting translation initiation. The inhibition of PKR has the opposite effect. We studied the effect of TAR on PKR by positioning TAR at a distance of the 5’ end where it cannot interfere with the binding of the 40S. Our results showed that a small amount of TAR, which activates PKR, increases the frameshift efficiency whereas a large amount of TAR, which inhibits PKR, decreases it. A model is presented where the variations of translation initiation modulate HIV-1 frameshift efficiency by altering the distance between the elongating ribosomes. This influences the probability that these ribosomes encounter or not a folded FSS. We next observed the effect of the 5’ untranslated region (UTR) of HIV-1 full length mRNA on its frameshift efficiency. This 5’UTR contains several structured parts, including TAR at the 5’end, which can inhibit translation initiation. This mRNA has a cap and an internal ribosome entry site (IRES) and could then use a cap dependent and an IRES-dependent mode of translation initiation. We replaced the 5’UTR of our bicistronic reporter mRNA by the complete 5’UTR of HIV-1 full-length mRNA or a part of it. Our results showed that the presence of the complete 5’UTR inhibits cap-dependent initiation of translation and increases the frameshift efficiency. Those effects are mostly due to the presence of TAR followed by a Poly(A) stem-loop. We also constructed a tricistronic reporter where the ribosomes translating the luciferases have to use an IRES-dependent initiation mode. The rate of this initiation was low and the frameshift efficiency obtained was also low. We proposed a hypothesis accounting for this situation. We also observed that when both initiation modes are available, the cap-dependent mode seems to be highly favored. Finally, we studied the impact of the Tat viral protein on translation initiation and frameshift efficiency. We showed that the presence of Tat increases translation initiation and decreases the frameshift efficiency. Those effects are more important when TAR is present at the 5’end of mRNA. We propose a model explaining the effects of Tat on translation initiation by the inhibition of PKR and by changes in the expression of cellular proteins that are able to unfold TAR. Our results allow us to better understand the mechanisms controlling HIV-1 frameshift, which will help in the development of drugs targeting the HIV-1 frameshift.