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

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.

The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

“…1A). A similar strategy has been successfully used in previous studies (12,15,16,32). In this context the translational activity of the HTLV-1 5=UTR was measured by using FLuc activity as the readout, while the RLuc reporter gene was translated by a capdependent mechanism and serves as an internal control.…”

“…8A), were transfected into HeLa cells. Even though HeLa cells are not the natural target of HTLV-1, they were selected for these assays since they are easy to handle and modify (see the RPS25 data presented below), efficiently transfected, known to support IRES activity from other retroviral mRNAs, including that of the HIV-1 IRES (12,14,15), and HeLa cell extracts enhance HTLV-1 IRES activity (Fig. 5B).…”

“…The RT-PCR assay was carried out using a SuperScript III one-step RT-PCR system with a Platinum Taq DNA polymerase kit (Invitrogen) according to the manufacturer's protocol, using 400 ng of total RNA and the primers p2anti (5=-TCTCTTCATAGCCTTATGCAGTTG-3=) and Pforluc (5=-CATGACTTCGAAAGTTTATGATC-3=) as previously described (15). The PCR assay was conducted using the primers p2anti and Pforluc, 400 ng of total DNA, and the GoTaq Green Master mix (Promega), according to the manufacturers' protocol.…”

“…A different study later showed that scanning can also take place on the HIV-1 5=UTR in cultured cells (13). More recently, the presence of an IRES within the 5=UTR of the HIV-1 mRNA has been reconfirmed (14)(15)(16)(17). Taken together, these reports suggest that both mechanisms (cap-and IRESdriven) are used by the HIV-1 mRNA to drive the synthesis of the viral Gag protein (8,9,18,19).…”

Translation initiation of the HIV-1 mRNA

“…86,87 The IRES within the 5 0 UTR has been evidenced in the laboratory adapted HIV-1 infectious recombinant proviral clone NL4.3, the CXCR4 (X4)-tropic primary isolate HIV-LAI and in the 5 0 UTR of viral RNA isolated from clinical samples. 86,88,89 The characterization of the HIV-1 IRES was done by using a series of bicistronic constructs that were verified to contain no cryptic promoter or spurious splice sites as they are often a caveat in this area of research 86,[88][89][90] In addition, translation of HIV-1 in a bicistronic context was shown to be resistant to picornaviral proteases that specifically inhibit cap-dependent translation initiation. 86,89,91 More recently, Monette et al have used transfection-infection experiments of DNA encoding a bicistronic RNA containing the HIV-1 5 0 UTR proving that its activity is not disrupted by poliovirus infection in 293 T cells.…”