ZIKV Life Cycle Inhibition by RC-101

To determine which stage of the ZIKV life cycle was inhibited by RC-101, a time-of-addition experiment was performed as previously described (51 (link)). Vero cells were infected with ZIKV (MOI, 0.1) for 1 h (0 to 1 h). RC-101 (40 μM) was incubated with the cells for 1 h before infection (−1 to 0 h), coadministration infection (0 to 1 h), and for 47 or 71 h postinfection (1 to 48/72 h) (Fig. 2A). To exclude a possible direct inactivating effect of RC-101, ZIKV (MOI, 2.5) was incubated with RC-101 (40 μM) at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells for 1 h. To confirm the inhibitory effect of RC-101 against ZIKV replication, BHK-21 cells were electroporated with the ZIKV replicon (SZ-WIV001; GenBank accession no. KU963796) and then incubated with RC-101. Renilla luciferase activity in the cell lysates was measured using the Rluc system (Promega, Madison, WI, USA) (52 (link)).