Figure 3

Mutations within the minimal RSE suggest that multiple regions of sequence and structure contribute to function. A. Steady-state RNA levels obtained by RNase protection assay of each point mutant. Point mutations within each of the predicted stem-loops resulted in a partial loss of stability relative to the wild-type viral RNA. A compensatory mutation (Comp3) of Mutant 3 that was predicted to reform the secondary structure did not recover wild-type levels. B. Structural diagram of the predicted secondary structure of the minimal RSE based on in vitro structure studies. The regions that were deleted (Δ1 and Δ2) and the stems that were mutated (Mut1-Mut3) are indicated. The boxes corresponding to each mutation indicate the sequence variation (highlighted region) while the structure displays the wild-type sequence. C. A 14 nucleotide (deletion 1) and a 12 nucleotide (deletion 2) single-stranded region of the minimal RSE were deleted and the level of viral RNA was assayed by RNase protection assay. Values are reported as a fraction of wild-type RNA. The deletions resulted in a partial loss of function. To determine whether spacing within the RNA was altered, sequence was added to the 5' or 3' end or at the deletion site. D. Diagram depicting the organization of the deletion constructs and the location of the sequence added back. Δ: The location of the deleted sequence is represented by a dotted line. Mix: The checked box represents deleted sequence that was scrambled and then added back at the deletion site. 5': The diagonally hatched box represents 10 nts of viral sequence added back to the 5' end of the minimal RSE sequence that contains the deletion. 3': The diagonally hatched box represents 10 nts of viral sequence added back to the 3' end of the minimal RSE sequence that contains the deletion. The added viral sequence is that which naturally lies just 5' or just 3' of the minimal RSE. The diagram is not to scale. Stars indicate a significant reduction compared to the wild-type virus (*: p < 0.01; **: p < 0.001; ***: p < 0.0001)