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 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.
de la Peña
M
M
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Camino de Vera 14, 46022 Valencia, Spain.
Navarro
B
B
Flores
R
R
D019659:Asteraceae / Q000821:virology; D001483:Base Sequence; D017795:Bromovirus / Q000235:genetics / Q000472:pathogenicity*; D002874:Chromosome Mapping; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010935:Plant Diseases / Q000821:virology; D016337:RNA, Catalytic / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*; D014772:Viroids / Q000472:pathogenicity*
D016337:RNA, Catalytic; D012367:RNA, Viral