Multiple pathways of reversion in viroids for conservation of structural elements.

From site-directed mutagenesis of potato spindle tuber viroid (PSTVd) it had been concluded earlier that the formation of a thermodynamically metastable structure containing hairpin II (HP II) is critical for infectivity. In order to differentiate between structural and sequence effects, in the present work base pairs in HP II were exchanged by site-directed double mutations without significant alterations in the native rod-like structure of PSTVd. The mutants were viable and genetically stable in the first generation, but one of the two mutations reverted to the wild-type nucleotide in the second generation. Single-site mutations in the stem of HP II, which had been described as revertants to the wild-type sequence earlier, were analysed with respect to the time course of reversion and the sequence variation during reversion. All replicating sequence variants were separated by gel electrophoretic techniques and the sequences and their relative frequencies were determined. From both types of studies it can be concluded (i) that HP II is a functional element in the (-)strand replication intermediate, generated due to sequential folding during synthesis, and that it is essential for template activity of (+)strand synthesis; (ii) that G:U pairs are tolerated transiently in (-)strand HP II; the lower stability of such a HP II is compensated by additional mutations outside HP II which suppress the competition of a rod-like structure; and (iii) that the reversions are generated spontaneously during (-)strand synthesis. Furthermore, the double-stranded structure of HP II is the essential element for short term replication of PSTVd but the exact sequence of the wild-type proves to be superior with regard to fitness and replicability of PSTVd.