Formation of Circular Satellite Tobacco Ringspot Virus RNA in Protoplasts Transiently Expressing the Linear RNA

Abstract

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L−(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L−(+)M into the corresponding circular form C−(+)M. We transiently expressed L−(+)M wild type and L−(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C−(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L−(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53–211 and 268–350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L−(+)M. The mutant L−(+)M was found to be extensively ligated to C−(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3′ terminal sequence. A trinucleotide in the 3′ region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C−(+)M formation. These results suggest that the 3′ stem contributes to the suitability of the small L−(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structurein vivo.