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 thermodynamicall y metastable structure containing hairpin II (HP II) is critical for i nfectivity. In order to differentiate between structural and sequence effects, in the present work base pairs in HP II were exchanged by sit e-directed double mutations without significant alterations in the nat ive rod-like structure of PSTVd. The mutants were viable and genetical ly stable in the first generation, but one of the two mutations revert ed to the wild-type nucleotide in the second generation. Single-site m utations in the stem of HP II, which had been described as revertants to the wild-type sequence earlier, were analysed with respect to the t ime course of reversion and the sequence variation during reversion. A ll replicating sequence variants were separated by gel electrophoretic techniques and the sequences and their relative frequencies were dete rmined. From both types of studies it can be concluded (i) that HP II is a functional element in the (-)strand replication intermediate, gen erated due to sequential folding during synthesis, and that it is esse ntial for template activity of (+)strand synthesis; (ii) that G:U pair s are tolerated transiently in (-)strand HP II; the lower stability of such a HP II is compensated by additional mutations outside HP II whi ch suppress the competition of a rod-like structure; and (iii) that th e reversions are generated spontaneously during (-)strand synthesis. F urthermore, the double-stranded structure of HP II is the essential el ement for short term replication of PSTVd but the exact sequence of th e wild-type proves to be superior with regard to fitness and replicabi lity of PSTVd.