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Les mécanismes d’initiation de la traduction de la polyprotéine Gag du Virus de l’Immunodéficience Humaine (VIH-1)

Abstract : The Human Immunodeficiency Virus (HIV) genomic RNA is multifunctional. It acts both as a genome that is packaged within virions and as messenger RNA translated to yield the Gag and Gag-Pol polyproteins. The translation of these proteins relies exclusively on the cellular translation machinery and is initiated through two mechanisms: the canonical cap-dependent initiation pathway and the use of internal ribosome entry sites (IRESes). HIV-1 has two IRESes, one located within the 5' UTR (5' UnTranslated Region) that is stimulated during the G2/M phase of the cell cycle, and the other embedded within the Gag polyprotein coding region. The later drives translation initiation from two AUG in frame and results in the production of the full-length Gag protein but also of an additional N-terminally truncated Gag isoform. Few things are known about this isoform, but the mutation of the second AUG causes a significant decrease in the rate of viral replication. The structural and functional conservation of Gag IRES among lentiviruses suggests an important role of this isoform and thus of the IRES in the viral cycle. Our work aims to understand at a molecular level the host-pathogen relationships in the translation of the viral messenger RNA. My work focused on the roles of the 40S ribosomal subunit and of the cellular helicase DDX3 in the translation initiation of Gag. During the first part of my Phd, I studied the interaction between the 40S ribosomal subunit and HIV-1Gag IRES. Following complementary approaches, we evidenced two distinct ribosome binding sites present close to the two the initiation sites of Gag. Then, we evaluated the effect of each 40S binding site deletion on Gag translation efficiency, both in vitro and in cellulo (in collaboration with the team of T. Ohlmann, CIRI-ENS-Lyon). Taken together, our results confirm the functional relevance of the two ribosomal binding sites to ensure optimal production of the two Gag isoforms. The second part of my Phd project aims to define the role of DDX3 in the translation initiation of Gag. DDX3 is a RNA DEAD-box helicase involved in many cellular processes such as cell cycle regulation and the innate immune response but also in all aspects of RNA metabolism such as transcription, splicing, mRNA nuclear export and translation. Recently DDX3 has been shown to favor HIV-1 Gag translation. To define its role, we first purified a recombinant form of the protein and performed kinetic experiments to analyze its biochemical properties. Contrary to what has been previously described, MBP-DDX3 displays a strictly RNA-dependent ATPase activity with kinetic constants similar to those displayed by its yeast counterpart Ded1p. We next evaluated MBP-DDX3 helicase activity towards RNA duplexes or RNA/DNA hybrids, with different length and single strand overhangs. Our preliminary results indicate that DDX3 alone is sufficient to enhance Gag translation in our in vitro system which paves the way to fine biochemistry experiments such as reconstruction of functional initiation complexes assembled onto Gag RNA and evaluation of its role on Gag RNA structure.
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Submitted on : Thursday, July 19, 2018 - 5:32:05 PM
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Melissa Ameur. Les mécanismes d’initiation de la traduction de la polyprotéine Gag du Virus de l’Immunodéficience Humaine (VIH-1). Biologie moléculaire. Université Sorbonne Paris Cité, 2016. Français. ⟨NNT : 2016USPCB125⟩. ⟨tel-01844836⟩



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