Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1

Abstract

Viroids are small non-coding parasitic RNAs that are able to infect their host plants systemically. This circular naked RNA makes use of host proteins to accomplish its proliferation. Here we analyze the specific binding of the tomato protein Virp1 to the terminal right domain of potato spindle tuber viroid RNA (PSTVd). We find that two asymmetric internal loops within the PSTVd (+) RNA, each composed of the sequence elements 5'-ACAGG and CUCUUCC-5', are responsible for the specific RNA-protein interaction. In view of the nucleotide composition we call this structural element an 'RY motif'. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an approximately 5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II

“…Previous yeast three-hybrid studies showed that tomato VirP1 binds to the right-terminal region of (+)-PSTVd (Gozmanova et al, 2003;Maniataki et al, 2003), and VirP1 was indeed shown to be critical for viroid infectivity. Because the rightterminal region is not important for PSTVd replication based on genetic evidence (Zhong et al, 2008) and it is not the Pol II-binding site, VirP1 may not be involved in replication.…”

Effect of citrus hosts on the generation, maintenance and evolutionary fate of genetic variability of citrus exocortis viroid

“…Sour orange was the only host in which the T R domain of CEVd populations remained fully conserved. This T R domain is believed to be involved in long-distance viroid transport (Hammond, 1994;Maniataki et al, 2003) mediated by the interaction of an 'RY' motif formed in this domain with a phloematic host protein (Gozmanova et al, 2003). When the CEVd concentration was determined in the trees described in this experiment, a higher accumulation was observed in the trifoliate orange leaves than in the sour orange ones.…”

“…These motifs are the TCRs present only in some genera of this family and whose functions remain unknown , hairpins (HP) I and II probably involved in replication processes (Ding & Itaya, 2007) and the recently identified 'RY' motif located in the T R domain which is thought to be involved in longdistance viroid transport within plants (Gozmanova et al, 2003).…”

Current status of viroid taxonomy

“…Because the TCR and TCH occupy the same relative position in several members of the family, they are assumed to play some as yet unknown functional role(s). Other motifs, including a so-called RY motif and a polypurine tract are conserved in members of several species in the family Pospiviroidae [28,41].…”