Rluc system

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)).

To determine which stage of the ZIKV cycle was blocked by each drug, a time-of-addition experiment was performed as previously described^{23 (link)}. Vero cells were infected with ZIKV strain H/PF/2013 at a multiplicity of infection of 0.8 for 1 h (0–1 h). Ouabain (10 μM) and digoxin (10 μM) were incubated with the cells at the following time points: pre-infection (−1 to 0 h), during infection (0–1 h), and for 47 h post-infection (1–48 h). To exclude a possible direct inactivating effect of the two drugs, viruses were incubated with each drug (1 μM) at 37 °C for 1 h, and the mixtures were diluted by 100-fold to infect Vero cells. Forty-eight hours later, virus titers were determined by performing a plaque assay.
To ensure the effectiveness of ouabain and digoxin in inhibiting ZIKV replication, Huh-7 cells were electroporated with the ZIKV replicon and then incubated with the indicated concentration of either drug, after which Renilla luciferase activity in the cell lysates was measured using the Rluc system (Promega, Madison, WI, USA).