Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs

Abstract

As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunviroidae adopt multibranched conformations occasionally stabilized by kissing-loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

Keywords: RNA silencing; catalytic RNAs; hammerhead ribozyme; small non-coding RNAs.

Figure 1

Structural features of viroids. Upper and middle panels, schemes of the characteristic rod-like secondary structures of the genomic RNAs of Potato spindle tuber viroid (PSTVd) and Hop stunt viroid (HSVd) respectively (family Pospiviroidae). The approximate location of the five structural domains – terminal left (T_L), pathogenic (P), central (C), variable (V), and terminal right (T_R) – is indicated, as well as that of the central conserved region (CCR), the terminal conserved region (TCR), and the terminal conserved hairpin (TCH). Lower panel, scheme of the multibranched secondary structure of the genomic RNA of Peach latent mosaic viroid (PLMVd; family Avsunviroidae), in which the sequences conserved in most natural hammerhead ribozymes are boxed with black and white backgrounds for the (+) and (−) polarities, respectively; the kissing-loop interaction is indicated with lines, and the characteristic 12-nt hairpin insertion of the reference variant containing the pathogenicity determinant of an extreme chlorosis (peach calico) is highlighted with blue color. For a more detailed representation of the PSTVd secondary structure see Figure 5.

Figure 2

Hairpin I structures of the five type species of the family Pospiviroidae. This element of secondary structure is formed by the upper CCR strand and a flanking inverted repeat of PSTVd, HSVd, CCCVd, ASSVd, and CbVd-1 (Coleus blumei viroid 1). Red fonts indicate conserved nucleotides in structurally similar positions. Continuous and broken lines represent Watson–Crick and non-canonical base-pairs, respectively. Notice that the variability preserves the overall structure of hairpin I, including the terminal palindromic tetraloop, the adjacent 3-bp stem, and the long stem. Left inset, hairpin I of the wild-type CEVd variant used to transform A. thaliana (notice two co-variations with respect to PSTVd at the basis of the long stem). Reproduced with permission from Gas et al. (2007).

Figure 3

Model for processing in vivo of the oligomeric (+) replicative intermediates of the family Pospiviroidae. The model predicts a kissing-loop interaction between the palindromic tetraloops of two consecutive hairpin I motifs (A), with their stems then forming a longer interstrand duplex (B). This double-stranded structure is the substrate for cleavage at specific positions in both strands (C). Following a second conformational switch, the resulting unit-length strands adopt the extended rod-like structure with loop E (in outlined fonts) and the adjacent bulged-U helix (D), which is the substrate for ligation (E). R and Y refer to purines and pyrimidines, respectively, the S-shaped line denotes the UV-induced cross-link, and white arrowheads mark the cleavage sites in the double-stranded structure and the ligation site in the extended conformation. Reproduced with permission from Gas et al. (2007).

Figure 4

Geometric classification of RNA base-pairing. The upper panel shows that each nucleotide base has three edges (Watson–Crick, Hoogsteen, and sugar) that can potentially form hydrogen bonds with one of the three edges of another base. Thus, each base is represented by a triangle and can potentially pair with up to three other bases. The interacting bases can pair with a cis or trans relative orientation of their glycosidic bonds; this is illustrated in the lower panels for the cis and trans orientations of nucleotides pairing at the Hoogsteen edge of one base and the sugar edge of the second base. In these base-pairs, the Watson–Crick edges of the interacting bases are available for further interactions – with other RNAs, proteins, or small molecules. The cross and circle in the triangle where the Hoogsteen and sugar edges meet indicate 5′ → 3′ and 3′ → 5′ orientations, respectively, of the sugar-phosphodiester backbones relative to the plane of the page. W–C, Watson–Crick edge; H, Hoogsteen edge; SE, sugar edge. Reproduced with permission from Zhong et al. (2006).

Figure 5

A genomic map of PSTVd loop motifs that are essential/critical for replication and systemic trafficking. Superscripts 1 and 2 refer to data from Zhong et al. (2006) and Zhong et al. (2007), respectively. R, replication; T, trafficking. Reproduced with permission from Zhong et al. (2008).

Figure 6

Secondary structure and tridimensional model proposed for most natural hammerhead ribozymes. (A) Schematic representation of a typical hammerhead structure (that of the plus strand of PLMVd) as originally formulated. Residues strictly or highly conserved in most natural hammerheads are on a black background. Arrow marks the self-cleavage site and dashes indicate Watson–Crick (and wobble) base-pairs. (B) Schematic representation of the same hammerhead structure according to X-ray crystallography and NMR data. The proposed tertiary interaction between loops 1 and 2 that facilitates catalytic activity in vivo, is denoted with a gray oval. Dashes indicate Watson–Crick (and wobble) base-pairs and dots non-canonical interactions. (C) Detailed 3D model of this hammerhead structure showing the interactions between loops 1 and 2 (in magenta). Residues in yellow form a local element of higher-order structure (the uridine turn).