EVIDENCE FOR AN INTERNAL RIBOSOME ENTRY SITE WITHIN THE 5' NON-TRANSLATED REGION OF TURNIP MOSAIC POTYVIRUS RNA

The genomic RNA of potyviruses has a characteristic 5' non-translated region (5'NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure an d thus its translation may not proceed in the same way as most cellula r mRNAs. To investigate the role of the 5'NTR during translation, vari ous derivatives of the turnip mosaic potyvirus (TuMV) leader were fuse d to the reporter gene beta-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit re ticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5' NTR, whether they were capped or not, were efficiently translated, whe reas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in exce ss over functional transcripts, translation was inhibited in a dose-de pendent manner. Similarly, transcripts synthesized from the reverse co mplement of the 5'NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop no rmally prevented translation of control GUS transcripts but when the v iral leader was positioned after it a significant level of GUS activit y was measured, whether the transcripts were capped or not. On the oth er hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes b ound to an internal site within the TuMV 5'NTR and then presumably sca nned the sequence for the initiator AUG.