In silico survey of the central conserved regions in viroids of the Pospiviroidae family for conserved asymmetric loop structures

Abstract

Viroids are the smallest replicative pathogens, consisting of RNA circles (∼300 nucleotides) that require host machinery to replicate. Structural RNA elements recruit these host factors. Currently, many of these structural elements and the nature of their interactions are unknown. All Pospiviroidae have homology in the central conserved region (CCR). The CCR of potato spindle tuber viroid (PSTVd) contains a sarcin/ricin domain (SRD), the only viroid structural element with an unequivocal replication role. We assumed that every member of this family uses this region to recruit host factors, and that each CCR has an SRD-like asymmetric loop within it. Potential SRD or SRD-like motifs were sought in the CCR of each Pospiviroidae member as follows. Motif location in each CCR was predicted with MUSCLE alignment and Vienna RNAfold. Viroid-specific models of SRD-like motifs were built by superimposing noncanonical base pairs and nucleotides on a model of an SRD. The RNA geometry search engine FR3D was then used to find nucleotide groups close to the geometry suggested by this superimposition. Atomic resolution structures were assembled using the molecular visualization program Chimera, and the stability of each motif was assessed with molecular dynamics (MD). Some models required a protonated cytosine. To be stable within a cell, the pK a of that cytosine must be shifted up. Constant pH-replica exchange MD analysis showed such a shift in the proposed structures. These data show that every Pospiviroidae member could form a motif that resembles an SRD in its CCR, and imply there could be undiscovered mimics of other RNA domains.

Advances in Viroid-Host Interactions

“…Studies with the closely related citrus exocortis viroid (CEVd) supported the involvement of this structural element in the ligation of linear viroid RNAs into circular forms during replication (see Section 4) (40), with a similar role proposed for the loop E conserved in all viroids of the genus Pospiviroid. Phylogenetic analysis and 3D modeling identified alternative structural elements, resembling loop E, in the CCR of other nuclear-replicating viroids that appeared to lack this structural element (41). Mutational studies followed by bioassays in planta and in protoplasts identified other loops and bulges in the most stable secondary structure of PSTVd with potential functional roles in its replication and/or trafficking (42) (Figure 3).…”

Progress in 50 years of viroid research—Molecular structure, pathogenicity, and host adaptation

“…53)-55) Furthermore, an in silico survey showed that members of the Pospiviroidae family can form a motif that resembles a sarcin/ricin domain or loop E in their CCR and implied that there could be undiscovered mimics of RNA domains to recruit host factors. 56) 1.3. Structural motifs regulating pathogenicity.…”

Sarcin/Ricin Domain RNA Retains Its Structure Better Than A-RNA in Adaptively Biased Molecular Dynamics Simulations

“…The symmetric loop E of the Escherichia coli 5S RNA uses two upper bricks without the extrahelical bases to sandwich noncanonical cWW pairs. Many nuclear-replicating plant viroids have an SRD in their central conserved control region, , while the other nuclear-replicating viroids are postulated to have a mimic of the SRD that has essentially the same backbone structure using a different set of noncanonical base pairs …”

“…19 Figure 1A explains the numbering system we use for the domain. 20 SRD's name derives from another occurrence in the ribosome at the translational GTPase binding site, 21−26 where the toxins α-sarcin 27 and ricin 28 dock. The structure of the domain has been described in several reviews.…”

“…Many nuclear-replicating plant viroids have an SRD in their central conserved control region, 17,31 while the other nuclearreplicating viroids are postulated to have a mimic of the SRD that has essentially the same backbone structure using a different set of noncanonical base pairs. 20 We simulate RNA motifs using molecular dynamics (MD) with an enhanced umbrella sampling technique called adaptively biased molecular dynamics (ABMD), 32 a culmination of a number of methods for sampling rare events. [33][34][35][36]36 ABMD is useful because it does not require preexisting knowledge of the system's free energy and has a parsimonious collection of preset tools to monitor the structure.…”