SLA′ is not necessary for ZIKV RNA replication. Cartoon diagrams of WT (A) SLA and (B) SLA′ with insets for the three sets of mutations that introduce G·U wobble base pairs into the lower and upper portion of the TSL (TSL-L [G·U], yellow and TSL-U [G·U], green), and base stem (BS [G·U], blue). (C) Schematic representation of the ZIKV subgenomic replicon system, which contains the Renilla luciferase (RLuc) gene in place of the majority of the ZIKV structural proteins. (D) Full-length capped WT, TSL-L (G·U), TSL-U (G·U), or BS (G·U) ZIKV subgenomic RLuc reporter replicon RNAs were electroporated into BHK-21 cells. Luciferase activity was measured at 2–72 h post-electroporation. The lower limit of detection is indicated (LLOD). Data are representative of three independent biological replicates with three technical replicates, and error bars represent standard deviation (SD) of the mean. Statistical significance was determined by multiple Student's t-tests, (*) P ≤ 0.1, (**) P ≤ 0.05. (E) Most stable predicted secondary structure of the last 70 nt at the 3′ terminus of the negative strand for the TSL-U (G·U), TSL-L (G·U), and BS (G·U) mutants, as constrained by normalized SHAPE reactivity data.
