Nucleotide Sequence and Secondary Structure of Citrus Exocortis and Chrysanthemum Stunt Viroid

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Abstract
The complete nucleotide sequence of citrus exocortis viroid (CEV) and chrysanthemum stunt viroid (CVS) was established, using the labeling in vitro and direct RNA sequencing methods and a new screening procedure for the rapid selection of suitable RNA fragments from limited digests. The covalently closed circular single-stranded viroid RNA consist of 371 (CEV) and 354 (CSV) nucleotides, respectively. As previously shown for potato spindle tuber viroid (PSTV, 359 nucleotides), CEV and CSV also contain a long polypurine sequence. Maximal base-pairing of the established CEV and CSV sequences results in an extended rod-like secondary structure similar to that previously established for PSTV and as predicted from detailed physicochemical studies of all these viroids. Although the 3 viroid species sequenced to date differ in size and nucleotide sequence, there is 60-73% homology between them. As does PSTV, CEV and CSV also contain conserved complementary sequences which are separated from each other in the native secondary structure. The resulting secondary hairpins, being formed and observed in vitro during the complex process of thermal denaturation of viroid RNA, appear to have a vital, although yet unknown, function in vivo. The possible origin and function of viroids are discussed on the basis of the characteristic structural features and of a considerable homology with U1a RNA found for a region highly conserved in the 3 viroids.