Structural and functional characterization of the SLA′ structure at the 3′ terminus of the Zika virus negative-strand intermediate

ZIKV NS5 binds to both SLA- and SLA′-containing RNAs. Electrophoretic mobility shift assays (EMSAs) were performed using ZIKV NS5 and (A) the first 163 nt of the ZIKV positive strand, or (B) the last 163 nt of the ZIKV negative strand. For each RNA, 2.5 pmol (150 ng) of RNA was incubated with increasing (twofold) amounts of NS5 ranging from 0 to 20 pmol (0–2 µg) and then analyzed via agarose gel electrophoresis. Molar ratios of NS5:RNA are indicated. Data shown are representative of four independent replicates. (C) Binding curves of NS5 to SLA- and SLA′-containing RNAs based on the bound RNA fraction from the EMSAs in A and B. Data for each RNA were fit to a sigmoidal four-parameter logistic curve model, with R² values of R² = 0.9962 and R² = 0.9987 for SLA and SLA′, respectively. The dashed lines indicate 95% confidence intervals.