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

被引:61
作者
BASSO, J
DALLAIRE, P
CHAREST, PJ
DEVANTIER, Y
LALIBERTE, JF
机构
[1] UNIV QUEBEC, INST ARMAND FRAPPIER, CTR RECH VIROL, LAVAL H7N 4Z3, PQ, CANADA
[2] FORESTRY CANADA, PETAWAWA NATL FORESTRY INST, MOLEC GENET & TISSUE CULTURE GRP, CHALK RIVER K0J 1J0, ON, CANADA
关键词
D O I
10.1099/0022-1317-75-11-3157
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5'NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene beta-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte 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, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement 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 normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5'NTR and then presumably scanned the sequence for the initiator AUG.
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页码:3157 / 3165
页数:9
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