A kissing-loop interaction in a hammerhead viroid RNA critical for its in vitro folding and in vivo viability

Abstract

Chrysanthemum chlorotic mottle viroid (CChMVd) RNA (398-401 nucleotides) can form hammerhead ribozymes that play a functional role in its replication through a rolling-circle mechanism. In contrast to most other viroids, which adopt rod-like or quasi-rod-like secondary structures of minimal free energy, the computer-predicted conformations of CChMVd and Peach latent mosaic viroid (PLMVd) RNAs are branched. Moreover, the covariations found in a number of natural CChMVd variants support that the same or a closely related conformation exists in vivo. Here we report that the CChMVd natural variability also supports that the branched conformation is additionally stabilized by a kissing-loop interaction resembling another one proposed in PLMVd from in vitro assays. Moreover, site-directed mutagenesis combined with bioassays and progeny analysis showed that: (1) single CChMVd mutants affecting the kissing loops had low or no infectivity at all, whereas infectivity was recovered in double mutants restoring the interaction; (2) mutations affecting the structure of the regions adjacent to the kissing loops reverted to wild type or led to rearranged stems, also supporting their interaction; and (3) the interchange between 4 nucleotides of each of the two kissing loops generated a viable CChMVd variant with eight mutations. PAGE analysis under denaturing and nondenaturing conditions revealed that the kissing-loop interaction determines proper in vitro folding of CChMVd RNA. Preservation of a similar kissing-loop interaction in two hammerhead viroids with an overall low sequence similarity suggests that it facilitates in vivo the adoption and stabilization of a compact folding critical for viroid viability.

Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm

“…However, we found that the minimal free-energy secondary structure predicted for GHVd RNA (Fig. 3A) displayed remarkable similarity to that of PLMVd (2,27,28). The secondary structure of PLMVd contains a long branched region stabilized by a kissing-loop pseudoknot and an extensively base-paired arm that encodes two self-cleavable hammerhead ribozymes active in the plus and minus strands, respectively (2,27,28).…”

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

“…Moreover, we have not detected by native PAGE any Mg 2+ -induced difference in the mobility of the mc ASBVd (À) RNA, while we have observed for it a slower mobility than for its (+) counterpart that neither is influenced by Mg 2+ . This latter result, particularly, argues against the existence of a kissing-loop interaction in the mc ASBVd (À) RNA because an interaction of this kind in CChMVd causes a faster mobility consistent with a more compact folding [39], in contrast with the results observed in the coelectrophoresis of the mc ASBVd (+) and (À) RNAs (Fig. 2b).…”

“…2a), without dismissing the coexistence of others with minor changes undetectable by non-denaturing PAGE. The lack of noticeable Mg 2+ effects did not favour the presence of tertiary interactions like the kissing loops reported in peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) [38,39].…”

Estudios de Patogenicidad de Viroides del Género Apscaviroide y Hostuviroide en cítricos.

“…Este tipo de interacciones tiene importancia biológica ya que una alteración de la secuencia de estas zonas provoca la perdida de infectividad como se ha descrito in vivo mediante experimentos de mutagénesis dirigida en CChMVd (Gago et al 2005).…”

“…Mutants were generated following a PCR-based protocol (Byrappa et al, 1995) with minor modifications (Gago et al, 2005). Briefly, 5 ng of a derivative of plasmid pBluescript II KS (+) containing the full-length viroid sequence were amplified with 250 ng each of pairs of adjacent and phosphorylated primers, complementary and homologous to different segments of the viroid sequence, in which appropriated changes were introduced to incorporate the desired mutations (Table 1).…”