The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

Abstract

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5′-untranslated region (5′-UTR) of the mouse mammary tumor virus (MMTV). The 5′-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5′-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5′-UTR was resistant to the addition of m7GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5′-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

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

“…RNA concentrations were determined spectrophotometri- (31). The RNA was translated in nucleasetreated RRL as previously described (32). The L-protease-RRL was diluted 1:5 or 1:10 in nuclease-free water (IDT) and added (to a final concentration of 3 and 6% [vol/vol]) to the fresh nuclease-treated 35% (vol/vol) RRL and preincubated for 15 min at 30°C, prior to the addition of the bicistronic mRNA.…”

“…Oocytes were isolated from Xenopus laevis ovarian fragments by manual dissection as previously described (32). Oocytes were incubated at 15°C for 24 h in standard Barth's solution supplemented with 10 IU of penicillin-streptomycin/liter and 2 mM pyruvate (32).…”

“…Oocytes were isolated from Xenopus laevis ovarian fragments by manual dissection as previously described (32). Oocytes were incubated at 15°C for 24 h in standard Barth's solution supplemented with 10 IU of penicillin-streptomycin/liter and 2 mM pyruvate (32). To evaluate the viral IRES activity in oocytes, 6.25 ng of in vitro-transcribed capped and polyadenylated RNA was microinjected into each oocyte with glass micropipettes calibrated to deliver a final volume of 50 nl (32,37).…”

“…Oocytes were incubated at 15°C for 24 h in standard Barth's solution supplemented with 10 IU of penicillin-streptomycin/liter and 2 mM pyruvate (32). To evaluate the viral IRES activity in oocytes, 6.25 ng of in vitro-transcribed capped and polyadenylated RNA was microinjected into each oocyte with glass micropipettes calibrated to deliver a final volume of 50 nl (32,37). When HeLa cell extracts were used, oocytes were first microinjected with mRNAs as described and 15 min later microinjected with 0.2 g of HeLa cell extracts.…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…Subsequent studies demonstrated that IRES elements drive internal initiation of translation in the RNA of all members of the Picornaviridae family (Bakhshesh et al, 2008;Borman and Jackson, 1992;Brown et al, 1991;Hinton and Crabb, 2001;Hinton et al, 2000;Kaku et al, 2002;Kuhn et al, 1990;Nateri et al, 2000;Sweeney et al, 2012;Willcocks et al, 2011;Yu et al, 2011b). The genome of other RNA viruses also contains IRES elements, as illustrated by hepacivirus, pestivirus, dicistrovirus, retrovirus Tsukiyama-Kohara et al, 1992;Vallejos et al, 2010;Wilson et al, 2000), as well as some RNA viruses infecting plants and protozoa (reviewed in Martinez-Salas et al, 2012). Although the presence of an IRES element is a common feature of all picornavirus RNAs, some differences affecting the organization of the 5 UTR, described below, are specific of each genus.…”