The 5′ and 3′ non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers, including internal ribosomal entry sites (IRESs) comprised in the 5′ leaders of many uncapped viral mRNAs. Blackcurrant reversion virus (BRV) has a genome composed of two uncapped, polyadenylated RNAs with relatively short 5′ NTRs, almost devoid of secondary structure. In this work, a role of the RNA2 5′ NTR in translation was studied by using mono- and dicistronic Photinus pyralis and Renilla reniformis luciferase reporter mRNAs in protoplasts of Nicotiana benthamiana. The RNA2 5′ leader was found to confer efficient in vivo translation compared with the control 5′ NTR, and each half of the BRV leader was essential for stimulatory function. Such efficient translational enhancement was mediated, at least in part, through an IRES mechanism. Multiple RNA2 5′ NTR regions, complementary to a fragment of plant 18S rRNA demonstrated previously to be accessible for intermolecular mRNA–rRNA interactions and conserved between eukaryotes, were shown to be important for efficient translation. Similar mRNA–rRNA base-pairing potential was also predicted for the 5′ leaders of other nepoviruses.
Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin