Browsing by Author "Sargueil, Bruno"

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    Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors
    (OXFORD UNIV PRESS, 2011) Vallejos, Maricarmen; Deforges, Jules; Plank, Terra Dawn M.; Letelier, Alejandro; Ramdohr, Pablo; Abraham, Christopher G.; Valiente Echeverria, Fernando; Kieft, Jeffrey S.; Sargueil, Bruno; Lopez Lastra, Marcelo
    The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used 'standard' chemical and enzymatic probes and an 'RNA SHAPE' analysis to model the structure of the HIV-1 5' leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5' leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.
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    Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1
    (PUBLIC LIBRARY SCIENCE, 2012) Vallejos, Maricarmen; Carvajal, Felipe; Pino, Karla; Navarrete, Camilo; Ferres, Marcela; Pablo Huidobro Toro, Juan; Sargueil, Bruno; Lopez Lastra, Marcelo
    The 5'untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.
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    Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein
    (2018) Cáceres, C. Joaquín; Angulo, Jenniffer; Lowy de la Torre, Fernando; Contreras, Nataly; Walters, Beth; Olivares, Eduardo; Allouche, Delphine; Merviel, Anne; Pino, Karla; Sargueil, Bruno; Thompson, Sunnie R.; López Lastra, Marcelo Andrés
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    Polypyrimidine tract-binding protein binds to the 5' untranslated region of the mouse mammary tumor virus mRNA. and stimulates cap-independent translation initiation
    (2016) Cáceres, Carlos J.; Contreras, Nataly; Angulo, Jenniffer; Vera Otarola, Jorge; Pino Ajenjo, Constanza; Llorian, Miriam; Ameur, Melissa; Lisboa, Francisco; Pino, Karla; López Lastra, Marcelo Andrés; Lowy de la Torre, Fernando; Sargueil, Bruno
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    Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site
    (2023) Angulo, Jenniffer; Caceres, C. Joaquin; Contreras, Nataly; Fernandez-Garcia, Leandro; Chamond, Nathalie; Ameur, Melissa; Sargueil, Bruno; Lopez-Lastra, Marcelo
    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.