The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their <i>in vivo</i> RNA conformations

Eggplant latent viroid (ELVd), like other members of family <i>Avsunviroidae</i>, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (−) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. <i>In silico</i> predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. <i>In vitro</i> examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. <i>In vitro</i> SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions <i>in silico</i>. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed <i>in vivo</i> most of the stems predicted <i>in silico</i> and <i>in vitro</i>, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5′ ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (−) strands <i>in vivo</i> at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family <i>Avsunviroidae</i>.