FIG. 6.

Mutational analysis of the 5′TR element. (A) Schematic representation of the mutant CTV:524/956, which contained nt 524 to 956 in the 3′ region of CTV-ΔCla333. The nucleotide sequence of the plus and minus strands of nt 719 to 776 in CTV:524/956 is shown as RNA sequence. Positions of stem-loops 1 and 2 (SL1 and SL2) are shown as dark lines below the negative strand sequence. (B) Diagrammatic representation of the putative stem-loop structures (SL1 and SL2) of the minus-strand RNA sequence as predicted by MFOLD and schematic outline of different mutations (substitutions, deletions, compensatory changes, and site-specific changes) introduced in SL1 and SL2. See the text for the details of mutations in SL1 and SL2. (C) Northern blot hybridization analysis of the total RNA isolated 3 and 4 days postinoculation from protoplasts transfected with the mutants outlined above. Accumulation of the genomic (g) and sgRNAs hybridized with 3′-plus- and minus-strand (left and right panels, respectively) RNA-specific riboprobes are shown.