Coding capacity determines in vivo accumulation of a defective RNA of clover yellow mosaic virus

Abstract

Naturally occurring defective RNAs (D RNAs) derived from the potexvirus clover yellow mosaic virus (CYMV) contain large internal deletions yet maintain a single open reading frame (ORF) representing the in-frame fusion of 5' and 3' terminal ORFs. Capped transcripts of the prototype 1.2-kb D RNA of CYMV were synthesized in vitro and used to inoculate broad bean plants. Progeny D RNA accumulated only if synthetic D RNA transcripts were coinoculated with CYMV RNA. Several experiments showed that helper-dependent accumulation of the D RNA in vivo depended on the maintenance of its encoded fusion ORF. (i) D RNAs with six-residue deletions introduced early in the fusion ORF accumulated, whereas those with four-residue out-of-frame deletions at the same sites were nonviable. (ii) Analysis of D RNAs containing termination codons at different locations showed that only the most 3' stop codon (maintaining over 93% of the fusion ORF) was permissive for D RNA accumulation. (iii) D RNAs with small in-frame deletions and insertions in their 3' coding regions were viable. (iv) Nonviable D RNAs containing disrupted fusion ORFs could not be complemented by the presence in the infection of a D RNA encoding a complete fusion ORF. Taken together, the results indicate that the process of translation, rather than the encoded product, modulates an event(s) which influences the propagation and/or accumulation of this RNA in vivo. This represents a unique requirement among plant virus D RNAs.