Mutational analysis does not support the existence of a putative tertiary structural element in the left terminal...

Comparative sequence analysis suggests that the left terminal domain of Potato spindle tuber viroid (PSTVd) and other large pospiviroids may assume a branched tertiary structure containing two pseudoknots. To search for evidence of such a structure in vivo, the nucleotide sequences proposed to interact were mutagenized, tomato seedlings were inoculated with mixtures of potentially infectious PSTVd RNA transcripts and the resulting progeny were screened for compensatory sequence changes. Positions 6-11 and 330-335 tolerated only limited sequence variation, and compensatory changes consistent with formation of an intact pseudoknot were observed in only two of the plants examined. No variation was detected at positions 14-16 or 29-31. Passage of selected variants in Rutgers tomato led to an increase in virulence only upon reversion to wild-type PSTVd_Intermediate. The ability of the left terminal domain to assume a branched conformation containing pseudoknots does not appear to be an important determinant of PSTVd fitness.