A cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES

et al. (2013) A cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES. PLoS ONE 8(2): e56962. doi:10.1371/journal.pone.0056962 A cis -Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES Fernando Valiente-Echeverra 0 Maricarmen Vallejos 0 Anne Monette 0 Karla Pino 0 Alejandro Letelier 0 J. Pablo Huidobro-Toro 0 Andrew J. Mouland 0 Marcelo Lo pez-Lastra 0 Eric Jan, University of British Columbia, Canada 0 1 Laboratorio de Virolog a Molecular, Instituto Milenio de Inmunolog a e Inmunoterapia, Centro de Investigaciones Me dicas, Escuela de Medicina, Pontificia Universidad Cato lica de Chile , Santiago, Chile, 2 HIV-1 Trafficking Laboratory , Lady Davis Institute at the Jewish General Hospital , Montre al, Que bec , Canada , 3 Department of Experimental Medicine, McGill University , Montreal, Quebec , Canada , 4 Department of Microbiology and Immunology, McGill University , Montreal, Quebec , Canada , 5 Departamento de Fisiolog a, Centro de Envejecimiento y Regeneracio n, CARE, Facultad de Ciencias Biol o gicas, Pontificia Universidad Cato lica de Chile , Santiago , Chile Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1. - Funding: FV-E and MV conducted this work as part of their PhD Thesis and were supported by a CONICYT doctoral fellowship, AM was supported by a predoctoral CIHR studentship, and AL, who also participated in this study as part of his PhD Thesis, was supported by a VRAID, PUC fellowship and by a CONICYT beca de extensio n. This study was supported by grants from the Fondo Nacional de Ciencia y Tecnologa del Gobierno de Chile (FONDECYT) 1090318, Proyecto P09/016-F de la Iniciativa Cientfica Milenio del Ministerio de Economa, Fomento y Turismo to MLL, partial support from FONDECYT 1110672 and Proyecto Basal CARE 12/2007 to JPH-T, and the Canadian Institutes of Health Research (CIHR, MOP-56974) to AJM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. In eukaryotes, the expression of proteins is frequently subject to regulation at the level of the initiation of mRNA translation [1,2]. Translation initiation is a stepwise process by which the 40S ribosomal subunit is recruited to the mRNA, and scans it in a 5939direction until the first initiation codon (AUG) is encountered. Recognition of the initiation codon by the migrating initiation complex leads to 80S ribosome assembly. The primary mechanism of initiation of protein synthesis involves the recognition of the 59 cap structure (m7GpppN, where N is any nucleotide) on the mRNAs by eukaryotic translation initiation factors (eIFs) that deliver the 40S ribosomal subunit [1,2]. Alternatively, an RNA structure termed the internal ribosome entry site (IRES) can drive 40S ribosomal subunit recruitment and positioning on the mRNA independently from the 59cap structure [3]. Translation initiation from the capped and polyadenylated unspliced mRNA of the human immunodeficiency virus type 1 (HIV-1) can occur through a canonical cap-dependent or by an alternative IRES-mediated mechanism (reviewed in [4,5]). The unspliced HIV-1 mRNA harbors two IRESes, the first of which is in the 59UTR (here referred to as the HIV-1 IRES) [6,7], and the second of which is within the gag open reading frame (the HIV-1 gag IRES) [8]. This observed redundancy in the possible mechanisms used to initiate translation of the unspliced HIV-1 mRNA, cap- or IRES-dependent, is conserved among primate lentiviruses suggesting that translation initiation of the unspliced mRNA is a key step during the viral life cycle [4]. Cap-independent initiation has been proposed to allow the viral mRNA to bypass the constraints of global cellular translation repression that normally target cap-dependent translation initiation [1,2,9]. The HIV-1 IRES drives viral structural protein synthesis during oxidative and osmotic stress [7,10], during G2/M of the cell cycle [6,11] and even when eIF4G and the poly(A) binding protein (PABP), two proteins that are critical for capdependent translation initiation, are cleaved by the viral protease [1215]. The molecular mechanisms that determine the function of the HIV-1 IRES element are not clearly understood. However, recent reports suggest that translation initiation driven by the HIV-1 IRES is to some extent modulated by cellular proteins [11,1618]. Evidence also suggests that the activity of the HIV-1 IRES is negatively influenced by cis-acting elements distinct from the 59cap structure or the 39poly(A) tail [7]. Brasey et al. (2003) reported that when in the context of a bicistronic mRNA, the gag open reading frame (ORF) negatively influences translation initiation driven from the HIV-1 59UTR, while Gendron et al. (2011) defined another region upstream of the primer binding site (PBS), the IRES negative element (IRENE), that also impacts on activity of the HIV-1 IRES. In this study we further explore the possibility that an RNA region downstream of the Gag initiation codon acts in cis- to modulate the translation of the full-length HIV-1 mRNA. In fact, several cis-acting RNA elements, distinct from the 59cap structure, the 39poly(A) tail, and IRENE, are known to regulate HIV-1 Gag protein expression. These cis-acting elements include amongst others, the cis-acting repressive sequence or inhibitory sequences (CRS/INS), from here on referred to generically as the INS elements [1924]. The INS elements, which are scattered throughout the gag, pol, and env regions of HIV-1 mRNA [1924], restrict expression of HIV-1 structural proteins by yet undefined mechanisms. A consensus sequence has not been defined for these elements, but in general INSs are characterized as AU-rich elements (AREs) [23,24]. One such element, known as the INS-1, found within the Matrix (p17) Gag coding region is reported to function as an inhibitor of cap-dependent translation initiation [19,24]. For example, when inserted in the context of a heterologous non-viral mRNA the INS-1 (...truncated)