A tobamovirus genome that contains an internal ribosome entry site functional in vitro.

Most eukaryotic mRNAs are translated by a "scanning ribosome" mechanism. We have found that unlike the type member of the genus Tobamovirus, translation of the 3'-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhov et al., 1993; 1994) occurred in vitro by an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translated in vitro: (i) "MP-CP-3'NTR" transcripts contained movement protein (MP) gene, CP gene and the 3'-nontranslated region of crTMV RNA. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamoviruses in vivo. (ii) "deltaNPT-CP" transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) "CP-GUS" transcripts contained the first CP gene and the gene of Escherichia coli beta-glucuronidase (GUS) at the 3'-proximal position. The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRES(CP)) promoting internal initiation of translation in vitro. Dicistronic IRES(CP), containing chimeric mRNAs with the 5'-terminal stem-loop structure preventing translation of the first gene (MP, deltaNPT, or CP), expressed the CP or GUS genes despite their 3'-proximal localization. The capacity of crTMV IRES(CP) for mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation. Two mutants were used to study structural elements of IRES(CP). It was concluded that integrity of IRES(CP) was essential for internal initiation. The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs.

The 3'-untranslated region of brome mosaic virus RNA does not enhance translation of capped mRNAs in vitro.

The translation enhancing ability of cis-acting 3'-terminal untranslated region (3'-UTR) of brome mosaic virus (BMV) was examined. Two chimeric mRNA constructs translated in rabbit reticulocyte lysates contained the BMV coat protein (CP) gene and NPTI gene, respectively. It was shown that the 3'-UTR of BMV RNA enhanced the translational efficiency of uncapped but not capped messages.

Effects of sequence elements in the potato virus X RNA 5' non-translated alpha beta-leader on its translation enhancing activity.

The 5' non-translated alpha beta-leader sequence of potato virus X RNA consists of two regions: the alpha sequence (41 nucleotides with no G) and the beta sequence (42 nucleotides upstream from AUG). The alpha beta-leader has been shown to enhance strongly the expression of adjacent genes in chimeric mRNAs. This phenomenon has been postulated to be due to the unpaired conformation of the 5'-terminal 30 nucleotides and/or to the presence within the alpha region of the CCACC pentanucleotide complementary to the 3'-terminal conserved structure of 18S rRNA. Different derivatives of alpha beta-leader have been constructed for use in determining the contribution of separate elements of the alpha beta sequence to translational enhancement. It was found that deletion of the alpha sequence large fragment which was supposed to be unfolded did not reduce the delta alpha beta-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the beta sequence resulted in a considerable increase in activity of the alpha-leader showing that the beta region was dispensable for translation. Disruption or 'masking' of CCACC led to inactivation of the alpha beta-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of alpha beta-leader.