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VIEW ARTICLE   |    DOI: 10.1094/MPMI-6-444

Translational Frameshifting by Barley Yellow Dwarf Virus RNA (PAV Serotype) in Escherichia coli and in Eukaryotic Cell-Free Extracts. Rong Di. Dept. of Plant Pathology, Iowa State University, Ames 50011 U.S.A. S. P. Dinesh-Kumar(2), and W. Allen Miller(1,2). (1)Dept. of Plant Pathology, and (2)Molecular, Cellular and Developmental Biology Program, Iowa State University, Ames 50011 U.S.A. MPMI 6:444-452. Accepted 22 March 1993. Copyright 1993 The American Phytopathological Society.

The open reading frame (39K ORF) at the 5 end of the genome of barley yellow dwarf virus, PAV serotype (BYDV-PAV), overlaps with a 60K ORF by 13 nucleotides. Several approaches were used to show that the 60K ORF (putative polymerase gene) is translated by a low-frequency frameshift event in which some ribosomes shift into the 60K ORF rather than terminate at the 39K ORF stop codon. A sequence encompassing this region of overlap induced minus one (--1) translational frameshifting in heterologous and native contexts. In Escherichia coli, with the alpha subunit of lacZ used as a reporter gene, the rate of frameshifting caused by the BYDV-PAV sequence was approximately 3%. Amino acid sequencing of the transframe protein confirmed that ribosomes slip into the --1 frame in the overlapping region which includes a consensus shifty heptanucleotide: GGGUUUU. In a wheat germ translation system, BYDV-PAV genomic RNA from virions frameshifted about twice as efficiently as full-length transcripts from a cDNA clone. Frameshifting in rabbit reticulocyte lysates was much lower for either template. The identity of the 99-kDa wheat germ translation product was verified as the transframe protein by immunoprecipitation with antibody specific for the 60K ORF. These results support our previous observations of frameshifting in protoplasts and illustrate a subtle molecular control mechanism between this pathogen and its host cells.

Additional Keywords: expression vectors, in vitro translation, protein sequencing, viral gene expression, luteovirus.