Translational frameshifting by barley yellow dwarf virus RNA

Barley yellow dwarf virus is a single-stranded RNA virus, consisting of several serotypes including the PAV serotype which is the focus of this dissertation. The 39K open reading frame (ORF) at the 5[superscript]\u27 end of the 5.7 kb (kilobase) genome overlaps with a downstream 60K ORF by 13 nucleotides. Several approaches were employed to demonstrate that the 60K ORF (putative polymerase gene) is expressed by an unusual minus one (-1) translational frameshifting mechanism. A sequence encompassing this overlap region induces frameshifting in heterologous and native contexts. The efficiency of frameshifting induced by the PAV sequence was approximately 3% in E. coli, using the [alpha] subunit of lacZ as a reporter gene. Amino acid sequencing of the transframe protein confirmed that ribosomes slip into the -1 frame in the overlapping region. In a wheat germ translation system under optimized salt conditions, the frameshifting rate of full-length PAV transcripts was 0.8 to 1.6%. The identity of the 99-kDa transframe translation product was verified by immunoprecipitation with antibody specific for the 60K ORF. The stop codon of the 39K ORF and the sequence downstream of frameshift site were found to play critical roles in PAV frameshifting. The sequence that includes the 6.7K ORF at 3[superscript]\u27 end of the PAV genome was discovered to be very important in frameshifting in vitro. It is proposed that the RNA sequence of 6.7K ORF stabilizes PAV RNA and enhances translational frameshifting with the aid of some unidentified plant factors