Mapping the molecular determinant of pathogenicity in a hammerhead viroid: A tetraloop within the in vivo branched RNA conformation

Abstract

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398–399 nt that can adopt hammerhead structures in both polarity strands. We have identified by Northern-blot hybridization a nonsymptomatic strain (CChMVd-NS) that protects against challenge inoculation with the symptomatic strain (CChMVd-S). Analysis of CChMVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the mutations observed in CChMVd-NS molecular variants were previously identified in CChMVd-S RNA, but others were never found in this RNA. When bioassayed in chrysanthemum, cDNA clones containing the CChMVd-NS specific mutations were infectious but nonsymptomatic. Site-directed mutagenesis showed that one of the CChMVd-NS-specific mutations, a UUUC → GAAA substitution, was sufficient to change the symptomatic phenotype into the nonsymptomatic one without altering the final accumulation level of the viroid RNA. The pathogenicity determinant–to our knowledge, a determinant of this class has not been described previously in hammerhead viroids–is located in a tetraloop of the computer-predicted branched conformation for CChMVd RNA. Analysis of the sequence heterogeneity found in CChMVd-S and -NS variants strongly supports the existence of such a conformation in vivo, showing that the rod-like or quasi-rod-like secondary structure is not a universal paradigm for viroids.