An Internal Ribosome Entry Segment Promotes Translation of the Simian Immunodeficiency Virus Genomic RNA

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.

The packaging signal of simian immunodeficiency virus is upstream of the major splice donor at a distance from the RNA cap site similar to that of human immunodeficiency virus types 1 and 2

“…There is evidence in the literature to suggest that some lentiviruses use internal ribosomal entry to initiate translation (Ohlmann et al, 2000;Buck et al, 2001). It is conceivable that this is the case for SIV and that the deletion has somehow altered the structural context of the IRES or that of the Gag initiation codon and, in doing so, disrupted the process.…”

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

“…IRES elements have now been identified within the simian immunodeficiency virus (SIV) and the human immunodeficiency virus type I (HIV-1) (Ohlmann et al 2000;Buck et al 2001;Waysbort et al 2001;Brasey et al 2003), HIV-2 (Herbreteau et al 2005), and more recently, feline immunodeficiency virus (FIV) (Camerini et al 2008) members of the lentivirus family, suggesting that translation of the retroviral genomic RNA is tightly controlled (Balvay et al 2007). The human immunodeficiency virus type 2 is a member of the lentivirus group of retroviruses and is, with HIV-1, the etiological agent of AIDS in humans (Bock and Markovitz 2001).…”

Retrovirus Translation Initiation: Issues and Hypotheses Derived from Study of HIV-1

“…Sequences from HIV-2, human T lymphotropic virus type 1 (HTLV-1), reticuloendotheliosis virus strain A (RevA), RSV, and MLV have been shown to be capable of directing IRES activity in reporter mRNA [4,8,13,24,25,39,49,58,83].…”

“…Another study used rapamycin to interrogate the 5′ UTR of the related primate retrovirus, SIV mac for evidence of internal initiation [58]. These experiments utilized an MLV-based retroviral vector that expressed plap in a 5′ cistron and neo in the 3′ cistron with the intergenic region containing the SIV 5′ UTR.…”