NS5 binding results in a change in SHAPE reactivity in the top loop of SLA and SLA′. ΔSHAPE reactivity plots for (A), the first 70 nt of the 5′ terminus of the positive strand and (C), the last 70 nt of the 3′ terminus of the negative-strand in the presence or absence of ZIKV NS5 (i.e., ΔSHAPE = NS5 + RNA reactivity − RNA only reactivity). Data are shown as the normalized change in SHAPE reactivity upon NS5 binding at each nucleotide position from four biological replicates, and error bars represent the SEM. Significant (>magnitude of the average ΔSHAPE reactivity) increases (red) and decreases (blue) in SHAPE reactivity upon NS5 binding are indicated. The baseline significance is indicated with a dashed line. Nucleotides 1–3 in A and 16–20 in C were omitted due to high background reactivity (light gray), while nucleotides 1–15 in C were bound by the primer (dark gray). (B,D) ΔSHAPE reactivity data from (A,C) mapped onto the predicted structures of (B) SLA and (D) SLA′. Tick marks represent 10 nt intervals. Insets represent the normalized SHAPE reactivities of the respective top loop regions in the absence and presence of NS5. Nucleotides with very low (≤0.2), low (0.2–0.4, blue), intermediate (0.4–0.85, orange), and high (≥0.85, red) normalized SHAPE reactivity are indicated in each inset.
