Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader

The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the g rowing number of mRNAs known to have a complex leader sequence. The 612-nuc leotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is never theless possible due to the ribosome shunt mechanism, by which ribosomes ar e directly transferred from a take-off site near the capped 5' end of the l eader to a landing site near its 3' end. There they resume scanning and rea ch the first long open reading frame. We investigated in detail how the mul tiple sORFs influence ribosome migration either via shunting or linear scan ning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt rout e. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning -inhibiting structure. In this leader as well as in the wild type leader, p roper translation and termination of sORF A was required for efficient shun t and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one- codon (start/stop) sORF was not functional. The results implicate that in C aMV, shunt-mediated translation requires reinitiation. The efficiency of th e shunt process is influenced by translational properties of the sORF.