ANALYSIS OF A TRANSLATIONAL ENHANCER UPSTREAM FROM THE COAT PROTEIN OPEN READING FRAME OF POTATO-VIRUS-S

Evidence has suggested that the subgenomic RNA of the carlavirus potat o virus S is an efficient message for the coat protein, even though ev idence suggests it is uncapped at its 5' terminus. We have investigate d 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 (vira l translational enhancer) was found to increase levels of translation in comparison to a synthetic leader when linked to the beta-glucuronid ase (GUS) reporter gene in vitro in rabbit reticulocyte and wheat germ lysate. VTE was also able to increase translation of the reporter gen e luciferase (LUC) in vitro above the levels obtained for both a synth etic leader and a leader obtained from a plant gene isolated from Arab idopsis thaliana. The level of enhancement was evident with both cappe d and uncapped transcripts. When the VTE sequence was deleted to 20 nu cleotides of the upstream region, thus removing the nucleotide block h omologous among carlaviruses, the ability to enhance levels of transla tion was removed. In vitro translation studies indicated that the tran slational enhancement activity of VTE was at least partially cap indep endent. 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 competitio n analysis revealed that when short RNA transcripts representing the 1 01 nucleotides of VTE were added in trans to functional VTE leader LUC constructs there was a marked decrease in the level of translation wh en compared with a synthetic leader added in trans. These results sugg est 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 VT E sequence.