Virus-like particle formation and translational start site choice of the plant retrotransposon Ttol

Ty 1/copia group retrotransposon T to 1 from tobacco was put under control of an inducible promoter for expression in Arabidopsis thaliana. The system was used to analyze intermediates of the transposition process. The T to 1 RNA 5' region has a complex structure and contains several AUG codons. We therefore sought to experimentally define the translation initiation site. Constructs starting at various positions within the structural gag region were expressed in planta and functionally characterized. We found that gag proteins starting at the first ATG of the gag-pol ORF (ATG1), but also those starting at the third ATG of the gag-pol ORF (ATG3), can form virus-like particles (VLPs). However, gag protein expressed by the inducible T to 1 element had a size similar to gag starting at ATG1, and mutation of ATG1 in the inducible element abolished reverse transcription. This suggested that translation initiation at ATG1 is essential for the T to 1 life cycle. To support this conjecture, gag protein starting at ATG1, or gag protein shortened amino-terminally by nine amino acids (starting at the second ATG of the gag region, ATG2), was coexpressed with T to 1 carrying mutations at ATG I and ATG2. Trans-complementation of the defective T to element by gag starting at ATG I, but not by gag starting at ATG2, defines ATG1 as the functional translation initiation site. (C) 2007 Elsevier Inc. All rights reserved.