Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region

Abstract

The RNA genome of tobacco etch virus (TEV), a plant potyvirus, functions as an mRNA for synthesis of a 346-kilodalton polyprotein that undergoes extensive proteolytic processing. The RNA lacks a normal 5' cap structure at its terminus, which suggests that the mechanism of translational initiation differs from that of a normal cellular mRNA. We have identified a translation-enhancing activity associated with the 144-nucleotide, 5' nontranslated region (NTR) of the TEV genome. When fused to a reporter gene encoding beta-glucuronidase (GUS), the 5' NTR results in an 8- to 21-fold enhancement over a synthetic 5' NTR in a transient-expression assay in protoplasts. A similar effect was observed when the 5' NTR-GUS fusions were expressed in transgenic plants. By using a cell-free translation system, the translation enhancement activity of the TEV 5' NTR was shown to be cap independent, whereas translation of GUS mRNA containing an artificial 5' NTR required the presence of a cap structure. Translation of GUS transcripts containing the TEV 5' NTR was relatively insensitive to the cap analog m7GTP, whereas translation of transcripts containing the artificial 5' NTR was highly sensitive. The 144-nucleotide TEV 5' NTR synthesized in vitro was shown to compete for factors that are required for protein synthesis in the cell-free translation reaction mix. Competition was not observed when a transcript representing the initial 81 nucleotides of the TEV 5' NTR was tested. These results support the hypothesis that the TEV 5' NTR promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to internal sites within the NTR.