Abstract
The retroviral genomic RNA is the messenger for the synthesis of the group-specific antigen (gag) and polymerase precursors of the major structural proteins and enzymes of the virion. The 5'-untranslated leader of the simian immunodeficiency virus (SIV) genomic RNA is formed of highly structured domains involved in key steps of the viral life cycle. Thus, the presence of stable RNA structures between the 5'-cap and the gag start codon are thought to strongly inhibit scanning of a 43 S preinitiation ribosomal complex. This prompted us to look for an alternative to the canonical ribosome scanning. By using a standard bicistronic assay in the rabbit reticulocyte lysate, we show that the SIVmac 5'-leader contains an internal ribosome entry segment (IRES) and that gene expression driven by this IRES is stimulated upon cleavage of eukaryotic initiation factor 4G. Deletion analysis revealed that the sequence between the major splice donor and the gag AUG codon is required for IRES activity. DNA transfection and viral transduction experiments in both NIH-3T3 and COS-7 cells confirmed that translation driven by the SIV leader is IRES-dependent and thus insensitive to the immunosuppressant rapamycin. Identification of an IRES in SIV is of particular interest for the understanding of lentivirus replication and also for the design of novel lentiviral vectors suitable for gene transfer.
Highlights
RNA-dependent helicase activity [4]; and eIF4G, which makes a bridge between the mRNA cap and the 40 S ribosomal subunit [5]
The 5Ј-Leader of simian immunodeficiency virus (SIV) Promotes Gene Expression when Inserted into a Bicistronic Construct—The complete SIV leader (Fig. 1), from positions 1 to 537 (numbering is with respect to the genomic RNA cap site), was inserted upstream of the lacZ reporter gene
Internal ribosome entry segments have been characterized in all picornaviruses identified to date [8], other RNA viruses (39 – 41), and some cellular mRNAs [42,43,44,45]
Summary
RNA-dependent helicase activity [4]; and eIF4G, which makes a bridge between the mRNA cap (via eIF4E) and the 40 S ribosomal subunit (via eIF3) [5]. An alternative mechanism of translation initiation has been described with the study of the picornavirus family This mechanism, called internal initiation, is rendered possible by an internal ribosome entry segment (IRES), which is a 450-nucleotide RNA sequence with complex secondary structures that allows translation by direct ribosome binding to the 5Ј-untranslated region (6 – 8). This has first been described by inserting specific cis-acting sequences into the intercistronic spacer of a bicistronic construct coding for two proteins [9, 10]. As reported for other IRESs, this expression was stimulated by the cleavage of eIF4G in vitro and upon treatment with the immunosuppressant rapamycin ex vivo
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