Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication

Abstract

RNA-templated RNA replication is essential for viral or viroid infectionAccording to the "RNA world" scenario, the appearance of RNA molecules simultaneously capable of self-replication and information storage signaled a major milestone in the evolution of life (14,24,25). In modern biology, RNA replication is central to viral or viroid infection, as well as to the regulation of cellular gene expression. Virus-encoded RNA-dependent RNA polymerases play a major role in the replication of RNA viruses (10, 23). Cellular RNA-dependent RNA polymerases generate double-stranded RNAs as triggers for RNA silencing (1). Intriguingly, the DNA-dependent cellular RNA polymerases can also transcribe at least two types of RNA templates: viroid RNAs (11,15,59) and the human hepatitis delta virus (HDV) RNA (28, 60). The replication of viroid and HDV RNAs raises the question of whether the DNA-templated transcription machinery also replicates other cellular RNAs yet to be identified. Elucidating the replication mechanisms of these infectious RNAs should help address this question of profound biological interest.Viroids are the smallest known nucleic acid-based infectious agents and self-replicating genetic units. Their "genomes" consist of single-stranded, circular RNAs ranging in size from 250 to 400 nucleotides (23). Viroids can replicate and spread throughout an infected plant, although they do not encode proteins, do not have encapsidation mechanisms, and do not require helper viruses. Furthermore, they cause devastating diseases by altering host gene expression and developmental processes (11,59). Evidently, viroid RNA genomes contain all of the sequence and structural information needed to mediate or trigger the various functions associated with infection. Potato spindle tuber viroid (PSTVd) is the type member of the family Pospiviroidae (8, 12). The PSTVd genome consists of 359 nucleotides and assumes a rod-shaped secondary structure in the native state (48) with five structural domains, as shown in Fig. 1A (26). This secondary structure, which is typical of viroids in the family Pospiviroidae, comprises many loops and bulges flanked by short Watson-Crick helices. Formation of this secondary structure is necessary for infection (62). During asymmetric rolling-circle replication of PSTVd (5), the plus circular strands serve as templates for the synthesis of concatemeric, linear minus strands, which then function as the replication intermediates for the synthesis of concatemeric, linear plus strands. These are subsequently cleaved into monomers and ligated into circular molecules (Fig. 1B). Without encoding proteins, PSTVd replicates in the nucleus of a host cell and

Viroid quasispecies revealed by deep sequencing

“…S1A) in all data sets except D o . Loop E is involved, among other processes, in processing of (C) replication intermediates to circles [for review see ref 9] Such a deletion would destroy the tertiary structure of this loop, 32 which is consistent with its presence only in (C) strands. It should be noted, however, that any change not supported by the corresponding mutation in the opposite strand has a higher chance of originating from either sequencing or mapping errors.…”

On the role of Hoogsteen:Hoogsteen interactions in RNA: Ab initio investigations of structures and energies

“…Thus, the so-called isostericity matrices and isodiscrepancy indices ), based on C19-C19 distances of the paired bases, along with some other geometrical parameters, have been used to classify base pairs within particular families into groups of base pairs that can substitute each other in sequences while maintaining the local 3D topology of RNA structures. The strong correlation between the principles of covariation with the concept of isostericity has not only been demonstrated in several studies Lescoute et al 2005;Zhong et al 2006), but this correlation has been extensively exploited in designing tools for structural and functional annotation (Yang et al 2003) such as Ribostral (Mokdad and Leontis 2006), ISFOLD (Mokdad and Frenkel 2008), and so on. Some issues related to the unambiguous assignment of isosteric subfamilies to H:H base pairs…”

Structural differences within the loop E motif imply alternative mechanisms of viroid processing

“…Based on a combination of comparative sequence analysis and NMR and X-ray crystal structures of other loop E motifs, Zhong et al (2006) have recently proposed that the loop E motif of PSTVd contains (1) an A261/G98 trans Hoogsteen/sugar edge pair, (2) a U260/C259 cis Hoogsteen/sugar edge pair, (3) a U260/A99 trans Watson-Crick/Hoogsteen edge pair, (4) an A258/A100 trans Hoogsteen/Hoogsteen edge pair, (5) an A101/U257 trans Hoogsteen/sugar edge pair, and (6) a C256/C102 cis Watson-Crick/Watson-Crick bifurcated pair. As shown in Figure 1B, the loop E-like motif of CVd-IIIb contains one additional base pair; i.e., the A101/U257 trans Hoogsteen/ sugar edge pair in PSTVd has been replaced by two GC pairs of unknown geometry.…”

“…For example, replacement of the bulged C residue at position 259 with a U results in a dramatic increase in the rate of accumulation in tobacco (Wassenegger et al 1996;Qi and Ding 2002); similarly, a U/A substitution at position 257 affecting a non-Watson-Crick AU pair leads to the appearance of an unusual ''flat top'' symptom in tomato (Qi and Ding 2003). Although the molecular mechanism(s) responsible for these effects remains to be determined, Zhong et al (2006) have recently published a detailed model of the tertiary structure of the PSTVd loop E motif, and this model is being used to guide additional mutational analysis.…”