Fig. 2.

Packaging ability and secondary structure prediction of S4 with mutations in the 3′ UTR. (a) Mutations in the S4 3′ UTR (S4.9–S4.14). The 2 nt substitution in mutant S4.14 is boxed. (b) Upper panel: detection of WT or mutant S4.9–S4.14 ssRNAs in the reverse transcription packaging assay by reverse transcription PCR yielding an 874 bp product (white arrows). Middle and lower panels: as described in Fig. 1. For consistency, each experiment was performed twice. (c) Detail of the secondary ssRNA structures of WT S4 (S4 of BTV-9) and mutants S4.11–S4.14 as predicted using RNAfold prediction software. The hairpin loop of the putative packaging motif for the WT segment (S4) is shown in green, the stem–loop in red and the flexible stretch in blue.