Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

et al. (2012) Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1. PLoS ONE 7(4): e35031. doi:10.1371/journal.pone.0035031 Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1 Maricarmen Vallejos 0 1 Felipe Carvajal 0 1 Karla Pino 0 1 Camilo Navarrete 0 1 Marcela Ferres 0 1 Juan Pablo Huidobro-Toro 0 1 Bruno Sargueil 0 1 Marcelo Lo pez-Lastra 0 1 Sung Key Jang, Pohang University of Science and Technology, Republic of Korea 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 Centro de Envejecimiento y Regeneracio n, CARE , Departamento de Fisiolog a, Facultad de Ciencias Biolo gicas, Pontificia Universidad Cato lica de Chile , Santiago , Chile , 3 Laboratorio de Infectolog a, Facultad de Medicina, Pontificia Universidad de Chile , Santiago, Chile, 4 Centre national de la recherche scientifique , Unite Mixte de Recherche 8015, Laboratoire de cristallographie et RMN Biologique, Universite Paris Descartes , Paris , France 1 A46G , C95U, A96C, A161C, A215G, G217A, G224A, A227C, C233U, G265A, C300U, A301G, A305U The 59untranslated 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 59UTRs 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 59UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 59UTR of some of the HIV-1 variants was enhanced relative to that observed for the 59UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 59UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-59UTR. 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. - Funding: This study was supported by the Fondo Nacional de Ciencia y Tecnologa del Gobierno de Chile (FONDECYT 1090318) and Proyecto P09/016-F, de la Iniciativa Cientfica Milenio del Ministerio de Economa, Fomento y Turismo to MLL, PFB 12/2007, grants from SIDACTION (AI20-1-01577) and the Agence Nationale de Recherche sur le Sida (ANRS 2011-11188) to BS, and partial support from FONDECYT 1110672 to JPH-T, grant Projet International de Cooperation Scientifique (PICS 5283) between the Centre National de la Recherche Scientifique (CNRS)-France and the Comisio n Nacional de Investigacio n Ciento fica y Tecnolo gica (CONICYT)-Chile to BS and MLL, and funding from the Programa de Doctorado en Ciencias Medicas, Escuela de Medicina de la Pontificia Universidad Cato lica de Chile (PMD 10/09) to MV. During her work on this project MV was supported by a CONICYT doctoral fellowship. Competing Interests: The authors have declared that no competing interests exist. Initiation of protein synthesis in the eukaryotic cell is a complex process that leads to the assembly of the 80S ribosome at the start codon of the mRNA [1,2,3]. In eukaryotic cells two mechanisms for recruiting and positioning ribosomes on the mRNA have evolved [1,2,3]. The primary mechanism 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. Upon recruitment to the mRNA, the 40S ribosomal subunit scans downstream along the 59 untranslated region (UTR) until the initiation codon is encountered. Alternatively, an RNA structure termed the internal ribosome entry site (IRES) can drive 40S ribosomal subunit recruitment and positioning on the mRNA either at or upstream of the start codon [2,4,5]. The 59 UTR of the full length or genomic RNA of the human immunodeficiency virus type-1 (HIV-1), prototype member of the lentivirus genus of the Retroviridae and the etiologic agent of AIDS, plays key roles during the viral life cycle [6]. The study of the mechanism of translation exhibited by HIV-1 genomic RNA revealed that the synthesis of the viral structural protein Gag can be initiated both through the canonical cap-dependent mechanism [7,8,9], or by the alternative IRES-dependent mechanism [9,10,11,12,13,14]. The capped and polyadenylated HIV-1 genomic RNA has been described to harbor two IRESes, the first within the mRNAs 59UTR (here referred to as the HIV-1 IRES) [10,14], and the second within the Gag open reading frame (referred to here as the HIV-1 gag IRES) [11,12,15]. The observed conservation of both cap- and IRES-dependent mechanisms of translation initiation among primate lentiviruses, coupled with the redundancy this provides, suggests that initiation of proteins synthesis is a key process during the viral life cycle [9,12,13,16]. Since its first characterization [10] a number of additional reports have addressed various aspects concerning the HIV-1 IRES [9,13,14,17,18,19,20]. One recognized caveat, however, of these and other reports concerning the mechanism of HIV-1 translation initiation is that findings are based upon the study of the 59UTR sequence recovered from only two infectious clones of HIV-1, namely pNL4.3 and pLAI [10,14,18,19]. The existence and relevance of IRES activity in the context of natural HIV-1 variants remains a matter of controversy and debate [7,16]. In this study we demonstrate that the 59UTRs of viral RNAs isolated from HIV-1 infected individuals show a capability to drive cap-independent translation initiation similar to that observed for the 59UTRs of clones pNL4.3 and pLAI [10,14,18,19]. The HIV-1 59UTR Isolated from Natural Viral Variants Drives Translation when in the Context of a Bicistronic mRNA At present, IRESes are defined solely by functional criteria [21,22]. The presence or absence of IRES activity within mRNAs of natural HIV-1 variants (VAR) was therefore evaluated using a bicistronic mRNA approach similar to that described by Brasey et al. (2003) and Gendron et al. (2011). To generate bicistronic VAR vectors (Fig. 1A), natural variant 59UTR sequences (nucleotides 1336 with respect to HIV-1 clone pNL4.3; AF 324493) were recovered from randomly pooled viral RNA extracts and cloned into a dual luciferase (dl) reporter construct containing an upstream Renilla luciferase gene (RLuc (...truncated)