A kissing-loop interaction in a hammerhead viroid RNA critical for its in vitro folding and in vivo viability.
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.
Gago
Selma
S
UPV-CSIC, Universidad Politécnica de Valencia, Avenida de los Naranjos, Valencia 46022, Spain.
De la Peña
Marcos
M
Flores
Ricardo
R
D019659:Asteraceae / Q000821:virology; D001483:Base Sequence; D017795:Bromovirus / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D014644:Genetic Variation; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D010942:Plant Viruses / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D016337:RNA, Catalytic; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D014772:Viroids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D018749:RNA, Plant; C427204:hammerhead ribozyme