Identification of the initiation codon of plum pox potyvirus genomic RNA

The expression of plum pox potyvirus (PPV) genomic RNA takes place through translation of its unique long and functional open reading frame (ORF) into a large polyprotein that undergoes extensive proteolytic processing. In this paper we show that the AUG recognized as the initiation codon of the PPV ORF by in vitro translation systems is the one found at nucleotide position 147, in spite of the presence at position 36 of an in-phase AUG that marks the start of the ORF. Deletion of a substantial part of the PPV 5' nontranslated region (5'-NTR), from nucleotide 19 to 101, does not impair the in vitro translation of PPV synthetic transcripts. By introduction of mutations that disrupt either of these two AUGs into a full-length PPV cDNA clone, it is shown that, while alteration of the first AUG does not have any effect on virus viability, growth, or symptom induction, destruction of the second renders the viral RNA noninfectious. This result indicates that the AUG employed in vivo is also the second. The hypothesis that this AUG could be recognized through a ribosomal internal entry mechanism has been tested in vitro using various bicistronic transcripts in which the PPV 5'-NTR was internally placed. The second cistron of these bicistronic RNAs was translated, but only at low levels, indicating that the PPV 5'-NTR is not able to drive in vitro an efficient internal entry of the ribosomes and suggesting that PPV RNA translation might proceed through a conventional leaky scanning mechanism.