Is Hammerhead Self-Cleavage Involved in the Replication of a Virusold in Vivo?

Abstract

The hammerhead self-cleavage reaction is generally considered to be involved in the in vivo production of (+) and (-) monomeric RNAs of the viroid-like, satellite RNA or virusoid of lucerne transient streak virus (vLTSV) from multimeric replicative intermediates, as part of the symmetrical rolling circle mechanism. To test this, three cDNA clones of vLTSV RNA, mutated at sites that inactivate in vitro self-cleavage of (-) RNA, were inoculated as excised plasmid cDNA inserts, together with helper virus LTSV, on Nicotiana clevelandii plants. As was predicted if hammerhead self-cleavage is involved in in vivo cleavage of (-) RNAs, high molecular weight (-) vLTSV RNAs were present in total RNA extracts from these mutant inoculated plants, but not in wild-type inoculated plants. Surprisingly however, the mutated virusoids also produced monomeric (-) RNAs in vivo. Sequence analysis of cDNA clones prepared from progeny virusold RNA revealed 8-20% of progeny contained reversions and pseudo-reversions of the introduced mutations. Hence, monomeric (-) RNAs were most likely produced by restoration of in vivo self-cleavage activity in the (-) RNA. Overall, the results indicate that mutations which disrupted self-cleavage in vitro also abolished the production of monomeric (-) RNAs in vivo and that hammerhead self-cleavage is involved in the cleavage of multimeric (-) RNAs to monomers. The observation that greater than 50% of the progeny cDNA clones generated from plants inoculated with mutant vLTSV contained base changes, compared to 20% from wild-type inoculated plants, may reflect an interesting adaptive response on the part of the mutated virusoids.