The translation initiation mechanisms of the Gag HIV-1 polyprotein

L'ARN génomique du Virus de l'Immunodéficience Humaine-1 (VIH-1) est multifonctionnel. Il constitue le génome encapsidé dans les virions et sert d'ARN messager pour la traduction des protéines virales Gag et Gag-Pol. La traduction de ces protéines dépend exclusivement de la machinerie traductionnelle cellulaire et est initiée par deux mécanismes différents : l'initiation canonique dépendante de la coiffe et l'initiation par entrée interne des ribosomes (IRES). Le VIH-1 présente deux IRES, l'un dans la région 5' non traduite (5'-UTR) qui est stimulé en phase G2/M du cycle cellulaire et l'autre dans la région codante de Gag. Ce dernier permet l'initiation de la traduction sur deux AUG en phase et conduit à la production de la protéine Gag pleine longueur mais également à la production d'une isoforme alternative de Gag, tronquée en région N-terminale. Le rôle de cette isoforme reste mal connu. Toutefois la mutation du second AUG chez VIH-1 et donc la suppression de la seconde isoforme de Gag provoque une diminution importante du taux de la réplication virale. La conservation structurelle et fonctionnelle de l'IRES Gag parmi les lentivirus suggère un rôle important de cette isoforme et de l'IRES gag dans le cycle viral. Nos travaux visent à comprendre à un niveau moléculaire les relations hôtes-pathogènes lors de la traduction des messagers viraux. Je me suis particulièrement intéressée aux rôles de la sous unité ribosomale 40S et de l'hélicase cellulaire DDX3 dans l'initiation de la traduction de la polyprotéine Gag du VIH-1. La première partie de ma thèse est consacrée à l'étude de l'interaction entre la sous unité ribosomale 40S et l'IRES gag du VIH-1. Par l'utilisation d'approches complémentaires, nous avons pu démontrer la présence de deux sites distincts de liaison au ribosome qui sont présents à proximité des deux codons d'initiation. Nous avons ensuite évalué à la fois in vitro et in cellulo (en collaboration avec l'équipe de T. Ohlmann, CIRI-ENS-Lyon) l'effet de la délétion de chacun des sites de liaison au 40S sur l'efficacité de traduction de la polyprotéine Gag. Nos résultats valident l'importance fonctionnelle des sites de liaison au ribosome pour une production optimale des deux isoformes de la polyprotéine Gag. La seconde partie de mon travail a consisté à définir le rôle de DDX3 dans l'initiation « coiffe-dépendante » de la traduction de la polyprotéine Gag. DDX3 est une hélicase à ARN à boîte DEAD impliquée dans de nombreux processus cellulaires tels que la régulation du cycle cellulaire et la réponse immunitaire innée mais également dans tous les aspects du métabolisme de l'ARN comme la transcription, l'épissage, l'export nucléaire ou encore la traduction. Plus récemment, il a été montré que DDX3 est nécessaire à la traduction de l'ARN génomique du VIH-1, cependant son rôle exact n'a pas encore été défini. Nous avons purifié une forme recombinante de la protéine en fusion avec la MBP (Maltose Binding Protein) et effectué des cinétiques enzymatiques afin de caractériser ses propriétés biochimiques. Contrairement à ce qui a été précédemment décrit, nos résultats montrent que DDX3 possède une activité ATPase strictement ARN-dépendante avec des constantes cinétiques similaires à celles de son homologue chez la levure, Ded1p. Nous avons également évalué l'activité hélicase de la protéine en présence de substrats de longueur et de nature variables (duplex ARN/ARN ou des hétéroduplex ADN/ARN). D'un point de vue fonctionnel, nous avons réalisé une première série d'expériences qui confirme la stimulation exercée par DDX3 sur la traduction de Gag in vitro. Ces résultats permettent d'envisager la caractérisation biochimique fine des interactions DDX3-ARN viral ainsi que de disséquer le rôle de DDX3 dans l'expression du génome viral.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

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

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.L'ARN génomique du Virus de l'Immunodéficience Humaine-1 (VIH-1) est multifonctionnel. Il constitue le génome encapsidé dans les virions et sert d'ARN messager pour la traduction des protéines virales Gag et Gag-Pol. La traduction de ces protéines dépend exclusivement de la machinerie traductionnelle cellulaire et est initiée par deux mécanismes différents : l'initiation canonique dépendante de la coiffe et l'initiation par entrée interne des ribosomes (IRES). Le VIH-1 présente deux IRES, l'un dans la région 5' non traduite (5'-UTR) qui est stimulé en phase G2/M du cycle cellulaire et l'autre dans la région codante de Gag. Ce dernier permet l'initiation de la traduction sur deux AUG en phase et conduit à la production de la protéine Gag pleine longueur mais également à la production d'une isoforme alternative de Gag, tronquée en région N-terminale. Le rôle de cette isoforme reste mal connu. Toutefois la mutation du second AUG chez VIH-1 et donc la suppression de la seconde isoforme de Gag provoque une diminution importante du taux de la réplication virale. La conservation structurelle et fonctionnelle de l'IRES Gag parmi les lentivirus suggère un rôle important de cette isoforme et de l'IRES gag dans le cycle viral. Nos travaux visent à comprendre à un niveau moléculaire les relations hôtes-pathogènes lors de la traduction des messagers viraux. Je me suis particulièrement intéressée aux rôles de la sous unité ribosomale 40S et de l'hélicase cellulaire DDX3 dans l'initiation de la traduction de la polyprotéine Gag du VIH-1. La première partie de ma thèse est consacrée à l'étude de l'interaction entre la sous unité ribosomale 40S et l'IRES gag du VIH-1. Par l'utilisation d'approches complémentaires, nous avons pu démontrer la présence de deux sites distincts de liaison au ribosome qui sont présents à proximité des deux codons d'initiation. Nous avons ensuite évalué à la fois in vitro et in cellulo (en collaboration avec l'équipe de T. Ohlmann, CIRI-ENS-Lyon) l'effet de la délétion de chacun des sites de liaison au 40S sur l'efficacité de traduction de la polyprotéine Gag. Nos résultats valident l'importance fonctionnelle des sites de liaison au ribosome pour une production optimale des deux isoformes de la polyprotéine Gag. La seconde partie de mon travail a consisté à définir le rôle de DDX3 dans l'initiation « coiffe-dépendante » de la traduction de la polyprotéine Gag. DDX3 est une hélicase à ARN à boîte DEAD impliquée dans de nombreux processus cellulaires tels que la régulation du cycle cellulaire et la réponse immunitaire innée mais également dans tous les aspects du métabolisme de l'ARN comme la transcription, l'épissage, l'export nucléaire ou encore la traduction. Plus récemment, il a été montré que DDX3 est nécessaire à la traduction de l'ARN génomique du VIH-1, cependant son rôle exact n'a pas encore été défini. Nous avons purifié une forme recombinante de la protéine en fusion avec la MBP (Maltose Binding Protein) et effectué des cinétiques enzymatiques afin de caractériser ses propriétés biochimiques. Contrairement à ce qui a été précédemment décrit, nos résultats montrent que DDX3 possède une activité ATPase strictement ARN-dépendante avec des constantes cinétiques similaires à celles de son homologue chez la levure, Ded1p. Nous avons également évalué l'activité hélicase de la protéine en présence de substrats de longueur et de nature variables (duplex ARN/ARN ou des hétéroduplex ADN/ARN). D'un point de vue fonctionnel, nous avons réalisé une première série d'expériences qui confirme la stimulation exercée par DDX3 sur la traduction de Gag in vitro. Ces résultats permettent d'envisager la caractérisation biochimique fine des interactions DDX3-ARN viral ainsi que de disséquer le rôle de DDX3 dans l'expression du génome viral

HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity

International audienceDEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N-and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions

Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame

International audienceIn the late phase of the HIV virus cycle, the full length unspliced genomic RNA is exported to the cytoplasm and serves as mRNA to translate the Gag and Gag-pol polyproteins. Three different translation initiation mechanisms responsible for Gag production have been described. However a rationale for the involvement of as many translation pathways in gRNA translation is yet to be defined. The Gag-IRES has the singularity to be located within the Gag open reading frame and to directly recruit the 40S ribosomal subunit. To further characterize this interaction, we first probed the Gag-IRES RNA structure. We then developed an innovative integrative modelling approach and propose a novel secondary structure model for the Gag-IRES. The minimal 40S ribosomal subunit binding site was further mapped using different assays. To our surprise, we found that at least two regions within Gag-IRES can independently recruit the ribosome. Next, we validated that these two regions influence Gag translation both in vitro and in cellulo. These binding sites are mostly unstructured and highly A-rich, such sequences have previously been shown to be sufficient to recruit the ribosome and to support an IRES function. A combination of biochemical and functional data give insight into the Gag-IRES molecular mechanism and provide compelling evidences for its importance. Hypothesis about its physiological role reflecting its conservation amongst primate lentiviruses are proposed

Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5′UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5′UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM’s ability to bind RNA, abrogated the protein’s capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1′s ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein’s capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein’s ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES

Differential binding and co-binding pattern of FOXA1 and FOXA3 and their relation to H3K4me3 in HepG2 cells revealed by ChIP-seq

BACKGROUND: The forkhead box/winged helix family members FOXA1, FOXA2, and FOXA3 are of high importance in development and specification of the hepatic linage and the continued expression of liver-specific genes. RESULTS: Here, we present a genome-wide location analysis of FOXA1 and FOXA3 binding sites in HepG2 cells through chromatin immunoprecipitation with detection by sequencing (ChIP-seq) studies and compare these with our previous results on FOXA2. We found that these factors often bind close to each other in different combinations and consecutive immunoprecipitation of chromatin for one and then a second factor (ChIP-reChIP) shows that this occurs in the same cell and on the same DNA molecule, suggestive of molecular interactions. Using co-immunoprecipitation, we further show that FOXA2 interacts with both FOXA1 and FOXA3 in vivo, while FOXA1 and FOXA3 do not appear to interact. Additionally, we detected diverse patterns of trimethylation of lysine 4 on histone H3 (H3K4me3) at transcriptional start sites and directionality of this modification at FOXA binding sites. Using the sequence reads at polymorphic positions, we were able to predict allele specific binding for FOXA1, FOXA3, and H3K4me3. Finally, several SNPs associated with diseases and quantitative traits were located in the enriched regions. CONCLUSIONS: We find that ChIP-seq can be used not only to create gene regulatory maps but also to predict molecular interactions and to inform on the mechanisms for common quantitative variation