Structural and functional characterization of the 5' region of subgenomic RNA5 of cucumber mosaic virus.
The uncapped and ORF-less subgenomic RNA5 is produced in subgroup II strains of cucumber mosaic virus (CMV), but not in subgroup I strains. Its initiation nucleotide (nt 1903) is in a 21 nt conserved sequence (Box1) that is absent in CMV subgroup I. Putative non-coding RNA structural elements surrounding Box1 in the plus and minus strand were identified in silico and by in vitro RNase probing. Four main stem-loop structures (SLM, SLL, SLK and SLJ) were identified between nt 1887 and 1999 of isolate R-CMV (subgroup II), with notable differences within SLM and SLL between the two strands. Mutation of a stem-loop within SLM, even when the predicted wild-type structure was maintained, showed significant reduction in RNA5 levels in planta. Three mutants containing 3-4 nt substitutions between positions -39 and +49 showed significantly reduced levels of RNA5, while another similar mutant at positions 80-83 had RNA5 levels comparable to wild-type. Deletion of Box1 resulted in similar levels of RNA3 and 4 as wild-type, while eliminating RNA5. Insertion of Box1 into a subgroup I isolate was not sufficient to produce RNA5. However, in a mutant with an additional 21 nt of R-CMV 3' of Box1 (positions -1 to +41), low levels of RNA5 were detected. Taken together, these results have identified regions of the viral genome responsible for RNA5 production and in addition provide strong evidence for the existence of newly identified conserved structural elements in the 5' part of the 3' untranslated region.
Thompson
Jeremy R
JR
Plant Virology Group, ICGEB Biosafety Outstation, Via Piovega 23, 31056 Ca' Tron di Roncade, Italy.
Buratti
Emanuele
E
Molecular Pathology Group, ICGEB, AREA Science Park, Padriciano 99, 34012 Trieste, Italy.
de Wispelaere
Mélissanne
M
INRA, Laboratoire de Biologie Cellulaire, UR501, INRA-Versailles, 78026 Versailles cedex, France.
Tepfer
Mark
M
Plant Virology Group, ICGEB Biosafety Outstation, Via Piovega 23, 31056 Ca' Tron di Roncade, Italy.
D001483:Base Sequence; D019295:Computational Biology; D017799:Cucumovirus / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion
D012367:RNA, Viral; D012260:Ribonucleases