Formation of circular satellite tobacco ringspot virus RNA in protoplasts transiently expressing the linear RNA.
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 structure in vivo.
Chay
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CA
Department of Plant Pathology, College of Agricultural and Environmental Sciences, University of California, Davis 95616, USA.
Guan
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Bruening
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D001483:Base Sequence; D017791:Geminiviridae / Q000235:genetics; D005822:Genetic Vectors; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017860:Nepovirus / Q000235:genetics* / Q000502:physiology; D009690:Nucleic Acid Conformation; D010947:Plants, Toxic; D011523:Protoplasts / Q000378:metabolism / Q000821:virology; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000079962:RNA, Circular; D019244:RNA, Satellite / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014026:Nicotiana / Q000378:metabolism / Q000821:virology*; D014779:Virus Replication
D000079962:RNA, Circular; D019244:RNA, Satellite; D012367:RNA, Viral; D012313:RNA