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Identification De Facteurs De Régulation Du VIH-1 Chez Les Macrophages Humains

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Identification De Facteurs De Régulation Du VIH-1 Chez Les Macrophages Humains Identification de facteurs de régulation du VIH-1 chez les macrophages humains Mémoire Yann Breton Maîtrise en microbiologie-immunologie Maître ès sciences (M. Sc.) Québec, Canada © Yann Breton, 2016 Identification de facteurs de régulation du VIH-1 chez les macrophages humains Mémoire Yann Breton Sous la direction de : Michel J. Tremblay, directeur de recherche Résumé Lors d'une exposition au VIH-1, bien qu'une seule petite proportion des macrophages soit infectée, il est proposé que ces cellules jouent un rôle important dans l'infection et la propagation du VIH-1. Pour approfondir nos connaissances dans ce domaine, des analyses transcriptomiques et protéomiques ont été effectuées afin de comparer les MDMs (Macrophages Dérivés de Monocytes) infectés aux non infectés. Ces analyses ont mené à la sélection de 50 gènes dont l'expression est modulée chez les cellules infectées pour effectuer un criblage par siRNA pour leurs rôles fonctionnels dans le cycle viral. Huit cibles ont été identifiées comme des régulateurs de l'infection chez les MDMs, mais seulement le gène MDM2 agissait comme un facteur de susceptibilité. Ce gène a donc été l'objet d'études plus approfondies. L'inhibition de l'expression de MDM2 induit une diminution de moitié de l'expression virale. Nos résultats indiquent que la résistance accrue au VIH-1 associée à l’interférence de MDM2 est maintenue même si le niveau d'ARNm est rétabli, suggérant que cette protéine serait impliquée indirectement dans l'infection par le VIH-1. L'identification des cofacteurs viraux régulés par MDM2 mènera à une compréhension des évènements signalétiques contrôlant la réplication du VIH-1 dans les macrophages. iii Abstract Upon exposure to HIV-1, only a small proportion of macrophages are infected whereas most remain uninfected. It is proposed that these cells play an important role in the establishment and propagation of HIV-1 infection. To further our knowledge in this field, transcriptomic and proteomic comparative analyses of uninfected and HIV-1-infected MDMs (Monocyte-derived macrophages) were performed. These analyses led to the selection of 50 genes that were tested for their functional roles in HIV-1 replication by siRNA screen. Eight genes were identified as regulators of HIV-1 infection in MDMs, but only MDM2 acted as a susceptibility factor. The knockdown of MDM2 decreased HIV-1 expression by two folds. Our results indicate that the resistance to HIV-1 upon MDM2 silencing is maintained in MDMs even if MDM2 mRNA level is restored, thus suggesting that this protein might be indirectly involved in HIV-1 infection. Identification of viral cofactors regulated by MDM2 will bring a new understanding of signaling events controlling HIV-1 replication in macrophages. iv Table des matières Résumé ................................................................................................................................. iii Abstract ................................................................................................................................. iv Table des matières ................................................................................................................. v Liste des tableaux ............................................................................................................... viii Liste des figures .................................................................................................................... ix Liste des abréviations ............................................................................................................ x Remerciements ................................................................................................................... xiii Chapitre 1: Introduction ........................................................................................................ 1 1.1. Le virus de l'immunodéficience humaine de type 1 ............................................. 1 1.1.1. Historique et état actuel de l'épidémie ........................................................... 1 1.1.2. Le génome ..................................................................................................... 2 1.1.3. Structure du virion ......................................................................................... 3 1.1.4. Cycle de réplication du VIH-1 ....................................................................... 4 1.1.4.1. Évènements précoces .............................................................................. 4 1.1.4.2. Évènements de biosynthèse .................................................................... 5 1.1.4.3. Évènements tardifs ................................................................................. 6 1.1.5. Immunopathogenèse du VIH-1 ..................................................................... 8 1.1.5.1. Transmission du virus ............................................................................. 8 1.1.5.2. Phases de l'infection ............................................................................... 9 1.1.5.3. Les réservoirs viraux ............................................................................ 11 1.1.6. Les antirétroviraux ....................................................................................... 12 1.2. Les macrophages ................................................................................................ 13 1.2.1. Les macrophages tissulaires ........................................................................ 13 1.2.2. Les macrophages dérivés de monocytes ...................................................... 14 1.2.3. La polarisation des macrophages ................................................................. 15 1.3. L'infection des macrophages par le VIH-1 ......................................................... 17 1.3.1. Caractéristiques du cycle viral chez les monocytes et macrophages ........... 17 1.3.2. Polarisation des macrophages et VIH-1 ...................................................... 19 1.3.3. Impacts du virus sur les fonctions cellulaires .............................................. 20 1.3.4. Les facteurs de restriction virale .................................................................. 20 Chapitre 2: Hypothèse et objectifs de recherche ................................................................. 24 Chapitre 3: Matériels et Méthodes....................................................................................... 25 v 3.1. Production de particules virales .......................................................................... 25 3.1.1. Modèles viraux ............................................................................................ 25 3.1.2. Culture de la lignée cellulaire 293T ............................................................ 26 3.1.3. Transfection au calcium-phosphate ............................................................. 27 3.1.4. Récolte et conservation de la production virale........................................... 27 3.1.5. Caractérisation de la production virale ........................................................ 28 3.1.5.1. ELISA de la protéine de capside p24 ................................................... 28 3.1.5.2. Culture de la lignée cellulaire TZM-bl et TCID50 des particules virales ................................................................................................................................. 29 3.2. Isolement et culture de MDMs à partir de sang périphérique de donneurs sains 30 3.3. Criblage de petits ARN interférents chez les MDMs ......................................... 32 3.3.1. Transfection des siRNAs ............................................................................. 32 3.3.2. Infection des MDMs et analyse de l'infection ............................................. 33 3.4. Validation des cibles potentielles ....................................................................... 34 3.4.1. Cytométrie de flux ....................................................................................... 34 3.4.2. Extraction d'ARN et PCR quantitative ........................................................ 35 3.4.3. Immunobuvardage ....................................................................................... 36 3.4.4. Cinétique de production virale .................................................................... 37 3.5. Analyses statistiques ........................................................................................... 37 Chapitre 4: Résultats ............................................................................................................ 38 4.1. Identification de protéines régulatrices du VIH-1 chez les macrophages humains ......................................................................................................................................... 38 4.1.1. Criblage de petits ARN interférents ............................................................ 38 4.1.2. Efficacité des petits ARN interférents ......................................................... 40 4.2. Validation de MDM2 comme facteur de régulation du VIH-1 .......................... 42 4.2.1. Le gène MDM2 est surexprimé dans les macrophages infectés .................. 42 4.2.2. La transfection des siRNAs contre MDM2 diminue l'infection par le virus NL4-3-Bal-IRES-HSA ................................................................................................ 43 4.2.3. La diminution du niveau de MDM2 n'induit pas de mort cellulaire ..........
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