Cell-based assay was performed as reported earlier in CoStar White 96-well plates19 (link). Briefly, plasmids expressing NS5B were co-transfected along with plasmids to express RIG-I, as well as two luciferases: the firefly luciferase driven from an interferon-β promoter and a Renilla luciferase driven from a thymidine kinase (TK) promoter. Compounds (10 μM) were added 4 h after plasmid transfection. All transfections were performed using Lipofectamine 2000 following manufacturer’s protocol. Luciferase activity was measured 16 h after addition of the compounds using DualGlo Luciferase assay system reagents (Promega) according to manufacturer’s protocol. Luminescence was detected using Synergy HT Multi-Mode microplate reader (Bio-Tek, USA). Ratio of firefly to Renilla luciferase values was converted to percentages and plotted using Microsoft Excel. Value obtained with DMSO was considered as 100%.
For RIG-I assay, cells were transfected with plasmids expressing RIG-I and the two luciferase reporters as mentioned above. 24 h after introduction of the plasmids, RIG-I agonist 3PdsR27 (a dsRNA of 27 base pairs with 5′ triphosphate, ref. 38 (link)) was transfected into the cells at a 50 nM. Luciferase activity was measured 16 h post transfection of RNA agonist as described above.
For RIG-I assay, cells were transfected with plasmids expressing RIG-I and the two luciferase reporters as mentioned above. 24 h after introduction of the plasmids, RIG-I agonist 3PdsR27 (a dsRNA of 27 base pairs with 5′ triphosphate, ref. 38 (link)) was transfected into the cells at a 50 nM. Luciferase activity was measured 16 h post transfection of RNA agonist as described above.