PEACH LATENT MOSAIC VIROID IS LOCKED BY A 2',5'-PHOSPHODIESTER BOND PRODUCED BY IN-VITRO SELF-LIGATION

Although some viroid-like satellite RNAs possess self-cleavage and sel f-ligation activities, we show that the peach latent mosaic viroid (PL MVd) is unique among all known viroids since it also has such activiti es. These catalytic activities should have important roles in the roll ing circle replication of PLMVd. According to this proposed mechanism, self-cleavage of the multimeric strands occurs via hammerhead structu res producing monomers possessing 2',3'-cyclic phosphate and 5'-hydrox yl termini. In the most stable predicted secondary structure for PLMVd these two extremities are juxtaposed, in order for self-ligation to o ccur. To establish the nature of the phosphodiester bond produced by s elf-ligation, we followed the classical procedure of complete enzymati c RNA hydrolysis coupled with thin layer chromatography fractionation. Using this procedure, we report that the self-ligation of PLMVd trans cripts produces almost exclusively the 2',5' isomer (>96%). Primer ext ension assays also revealed that reverse transcriptase can read throug th this 2',5' linkage, suggesting that it does not prevent further rep lication of the viroid. Moreover, we have observed that this 2',5' lin kage is resistant to the debranching activity contained in nuclear ext racts, as well as being capable of preventing further viroid self-clea vage. Thus, if viroids do indeed self-ligate in vivo, the resulting 2' ,5'-phosphodiester bond could contribute to the stability of these RNA species. Finally, an analysis of both the sequence and the structural requirements for hammerhead self-cleavage and self-ligation suggests that these two RNA processes may be interrelated. We hypothesize that the intramolecular self-ligation which produces circular conformers ma y contribute to the circularization step of the rolling circle replica tion, while the intermolecular non-enzymatic ligation is a potential m echanism for the sequence reassortment of viroids and viroid-like spec ies. (C) 1997 Academic Press Limited.