Figure 3.

ZIKV^mini RNA is circularized. (A) Schematic illustration of the ZIKV^mini mutants examined. The dark and light purple represent the 5′ CS and 3′ CS, respectively. The black dotted line represents cyclization potential and the red bar represents the anti-sense oligo (ASO). (B and C) Native PAGE analysis of (B) ZIKV^mini containing mutations in either the CS or UAR elements and (C) WT ZIKV^mini after heating and snap-cooling in the presence or absence of ASO or scrambled oligo (SCR). (D and E) SHAPE reactivity in the 5′ CS of the WT and mutant ZIKV^mini RNA. Nucleotides within the 5′ CS are enclosed by dotted brackets. WT SHAPE reactivity in light green is compared to (D) CS swap (dark green) and Δ3′ CS (purple) or to (E) WT annealed to SCR (dark green) or ASO (purple) RNA. Data are mean ± SD of normalized SHAPE reactivities at each base from three experiments. The same WT dataset is used in each panel as all conditions were examined simultaneously. SHAPE reactivities in the 5′ CS region of the WT and mutant minigenomes were compared by the Student's t-test. P-values are shown in Supplementary Table S2. (F) Heat map of SHAPE reactivities from a representative experiment overlaid onto ZIKV^mini (left/green) and ZIKV^mini-Δ3′ CS (right/purple) 5′ cyclization elements. The colour intensity represents increasing SHAPE reactivity as indicated in the box. See also Supplementary Figure S2.