Abstract
An infectious, in vitro transcript from a full-length cDNA clone of the barley yellow dwarf virus (PAV serotype) genome translated efficiently in a wheat germ translation extract. Deletions in a region that we call the 3' translational enhancer, located between bases 4,513 and 5,009 in the 5,677-base genome, reduced translation of the 5'-proximal open reading frames from uncapped RNA by at least 30-fold. Deletions elsewhere in all but the 5' end of the genome had no effect on translation. Presence of a m7G(5')pp(5')G cap on the 5' end fully restored translational efficiency of transcripts lacking the 3' translational enhancer. The translation enhancer reduced inhibition of translation by free cap analog, did not affect RNA stability, and did not function in reticulocyte lysates. When placed in the 3'-untranslated region of uncapped mRNA encoding the beta-glucuronidase gene, the translation enhancer stimulated translation more than 80-fold, in the presence of the viral, but not a plasmid-derived, 5' leader. A polyadenylate tail could not substitute for the 3' translation enhancer. These observations provide an extreme example, in terms of distance from the 5' end and level of stimulation, of an mRNA in which a sequence near the 3' end stimulates translation.
Highlights
From the Plant Pathology Department and Molecular, Cellular and Developmental Biology Program, Iowa State University, Ames, Iowa 50011-1020
The genes in the 5' -half of the genome are translated directly from genomic RNA, whereas the 3'-halfis expressed via subgenomic mRNA(s) [5, 8, 9]
The luteoviruses, including the barley yellow dwarf viruses (BYDVS),1 have been classified into subgroups I and II, according to genome organization and other biological properties 0, 2,10,11)
Summary
Vol 270, No 22, Issue of June 2, pp. 13446-13452, 1995 Printed in U.S.A. Yellow Dwarf Virus (PAV) Genome Strongly Stimulates Translation of Uncapped mRNA*. The 5'-half of the genome of BYDV-PAV, a member of subgroup I, encodes a 39-kDa protein (39K ORF) and an overlapping open reading frame (60K ORF) which has the conserved amino acid motifs shared by RNA-dependent RNA polymerases [6] This 60K ORF is expressed via a -1 ribosomal frameshift at the end of the 39K ORF to produce a 99-kDa protein (Fig. lA) [3, 4]. A sequence of at most 500 bases, located between 4.5 and 5 kilobases from the 5' end of the viral genome, stimulates translation of viral or heterologous reporter genes from uncapped RNA by 30- to over 100-fold. This cap-independent translation may depend on the presence of a portion of the 5' end of the viral genome. This report should further our understanding of interactions between mRNAs and eukaryotic translation machinery
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