Analysis of a translational enhancer upstream from the coat protein open reading frame of potato virus S

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Abstract
Evidence has suggested that the subgenomic RNA of the carlavirus potato virus S is an efficient message for the coat protein, even though evidence suggests it is uncapped at its 5′ terminus. We have investigated the effect of the upstream region of the coat protein gene of potato virus S on the level of reporter gene expression in vitro. The region of 101 nucleotides upstream of the coat protein, designated VTE (viral translational enhancer) was found to increase levels of translation in comparison to a synthetic leader when linked to the β-glucuronidase (GUS) reporter gene in vitro in rabbit reticulocyte and wheat germ lysate. VTE was also able to increase translation of the reporter gene luciferase (LUC) in vitro above the levels obtained for both a synthetic leader and a leader obtained from a plant gene isolated fromArabidopsis thaliana. The level of enhancement was evident with both capped and uncapped transcripts. When the VTE sequence was deleted to 20 nucleotides of the upstream region, thus removing the nucleotide block homologous among carlaviruses, the ability to enhance levels of translation was removed. In vitro translation studies indicated that the translational enhancement activity of VTE was at least partially cap independent. Translation of VTE linked to reporter genes in the presence of cap analogue was relatively unaffected whereas synthetic leader and a plant leader constructs were both more sensitive. In vitro competition analysis revealed that when short RNA transcripts representing the 101 nucleotides of VTE were added in trans to functional VTE leader LUC constructs there was a marked decrease in the level of translation when compared with a synthetic leader added in trans. These results suggest that the upstream region of the coat protein ORF of potato virus S promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to the 101 nucleotides of the VTE sequence.