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

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398-399 nt that can adopt hammerhead structures in both polarity strands. We have identifi ed 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, b ut others were never found in this RNA. When bioassayed in chrysanthemum, c DNA 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-t o our knowledge, a determinant of this class has not been described previou sly in hammerhead viroids-is located in a tetraloop of the computer-predict ed branched conformation for CChMVd RNA. Analysis of the sequence heterogen eity 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 se condary structure is not a universal paradigm for viroids.