A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation

TLDR

Picornavirus RNAs are uncapped and possess unusually long 5′ nontranslated regions rich in noninitiating AUGs, yet their translational efficiency varies markedly between cell‑free extracts. The study aimed to determine whether the 5′ NTR of encephalomyocarditis virus harbors an internal ribosomal entry site. Synthetic mono‑ and dicistronic mRNAs containing the EMCV 5′ NTR were generated by T7 RNA polymerase and translated in micrococcal nuclease‑treated HeLa cell extracts and rabbit reticulocyte lysates. The nucleotides spanning positions 260–484 of the EMCV 5′ NTR were essential for efficient translation in both mono‑ and dicistronic contexts, indicating that this region contains an IRES.

Abstract

Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.