Abstract
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.
Highlights
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 common with previous studies [10,18,19,23,24], the firefly luciferase gene (FLuc)/Renilla luciferase gene (RLuc) ratio was used as the readout of internal ribosome entry site (IRES) activity expressed as relative translation activity (RTA), with the mean translation efficiency of the reference IRES arbitrarily set at 100% (+/standard deviation)
These observations are in concordance with reported cap-independent translational activity by 59UTR sequences of other members of the Retroviridae and some retroelements [10,14,23,26,27,28,29,30,31,32,33,34,35,36,37], and strongly support the notion that the 59UTRs of all natural variants of human immunodeficiency virus type-1 (HIV-1) recovered from clinical samples harbor active IRES elements
Summary
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]. 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 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 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]
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