Figure 8. RNA secondary structure appears altered in subgenomic fragments containing disrupted pPSs.

In vitro transcripts of the ~1.5 kb region encompassing SL5877 and SL6067 were synthesised from both JFH-1 and mutant templates. RNA was folded and subjected to structural analysis via SHAPE mapping. Following NMIA (+) or DMSO (−) treatment and reverse transcription, a primer designed to bind approximately 20 nucleotides downstream of SL5877 was used for primer extension. Products were resolved on a 7% denaturing polyacrylamide gel (upper panel) and the relative reactivity was quantified using SAFA⁵⁸. Sequencing ladders were included to identify individual nucleotides, based upon the wildtype genome sequence.