Forced evolution reveals the importance of short open reading frame A and secondary structure in the cauliflower mosaic virus 35S RNA leader

Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants, In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF, Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.