Summary

Viroids are small, circular, single-stranded, non-protein-coding RNAs (246-401 nt) from plants. Despite these singular properties, viroids can replicate autonomously, move systemically, and in most cases cause disease in their hosts. Given that they do not encode proteins, replication of viroids relies very strictly on their interaction with host factors. This replication occurs via a rolling-circle mechanism based on RNA intermediates with three steps, which with some variations are repeated in the strands of both polarities: (1) RNA-RNA reiterative transcription of the initial circular template that generates oligomeric RNAs of complementary polarity, (2) cleavage of some of these RNAs to linear monomers, and (3) circularization of the latter. This Doctoral Thesis has focused on the study of the host factors involved in the third step of viroid replication.
For the identification of cellular factors involved in ligation of nuclear viroids (family Pospiviroidae), circularization of the linear monomeric RNA of Potato spindle tuber viroid (PSTVd), opened at the physiological processing site and with 5'-phosphomonoester (5'-P) and 3'-hydroxyl (3'-OH) termini, was assayed in the presence of protein chromatographic fractions of tomato, an experimental host of this viroid. An enzymatic activity able to circularize the viroid RNA was detected in fractions of the heparin column eluting around 0.5 M KCl. This ligation activity is specific of the RNA termini assayed and, therefore, different from the only plant RNA ligase known: the tRNA ligase, which recognizes 5'-hydroxyl (5'-OH) and 2',3'-cyclic phosphodiester (2',3'>P) termini. The new tomato activity is sensitive to thermal denaturation and proteolytic digestion, requires ATP and Mg2+, and shows high substrate specificity, circularizing only the linear PSTVd opened at the physiological processing site. The activity also mediates circularization of the linear monomeric RNAs of representative members of other genera of the family Pospiviroidae, all opened at their physiological processing sites. By using [a-32P]ATP in the ligation reaction, an adenylated protein of approximately 90 kDa with a chromatographic behavior identical to the RNA ligase activity was detected. Mass spectrometry analysis of a fraction containing this protein strongly suggested that the activity mediating circularization of the viroid RNA probably resides in the tomato DNA ligase 1. This hypothesis was confirmed when the corresponding cDNA was cloned and expressed in Escherichia coli, and the resulting protein was purified and tested in vitro using the linear PSTVd opened at the physiological processing site as substrate.
Regarding the host factors involved in the circularization of the replicative intermediates of chloroplastic viroids (family Avsunviroidae), it has been recently reported that the tRNA ligase may contain a transit peptide to the chloroplast probably resulting from a mechanism of alternative translation. This enzyme specifically recognizes 5'-OH and 2',3'>P RNA termini, which are those characteristically produced by the hammerhead ribozymes that mediate self-cleavage in the family Avsunviroidae. Consequently, the cDNA corresponding to the chloroplastic isoform of the eggplant tRNA ligase was cloned and expressed in E. coli. In vitro ligation assays with the purified recombinant protein, using the monomeric linear RNA of the Eggplant latent viroid (ELVd) with 5'-OH and 2',3'>P termini as substrate, showed that the tRNA ligase, in an ATP- and Mg2+-dependent reaction, efficiently catalyzes the circularization of the linear ELVd RNA only when it is opened at the physiological processing site. The enzyme was also able to circularize the monomeric linear RNAs of both polarities of the other chloroplastic viroids, indicating that most likely it is the host factor involved in the last step of their replication.