Substitutions in the stem of SL6. Three mutants were designed to investigate the influence of SL6 stem length. Most attempts to design synonymous substitutions had little effect on the stem folding conformation. Only silent mutant C33 (C 253 !A and C 255 !G) exhibited a significantly shortened duplex stem, with a corresponding elevated level of dG folding energy. These positions are highly conserved among the mTBFV (Figure 3 ) and, as expected, had a profound effect on virus replication; C33 displayed pinpoint plaques, reduced growth characteristics and almost no cpe (Table 1 and Figure 5 ).