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

摘要

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398-399 nt that can adopt hammer- head 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 CCh- MVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the muta- tions observed in CChMVd-NS molecular variants were pre- viously 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 mutagen- esis 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 heterogencity 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 struc- ture is not a universal paradigm for viroids.