Orion microplate luminometer

Generation of the WT EEEV and 11337 translation reporters were described previously [4 (link)]. The mutant EEEV translation reporters containing three-point mutations in either or multiple miR-142-3p binding sites in the 3’ UTR to eliminate miR-142-3p binding were generated singly or in combination using the QuikChange Mutagenesis II XL kit and the primers listed in S1 Table. A host mimic Firefly reporter RNA with short 5’ and 3’ UTRs was used as a control [4 (link)]. Electroporation was performed as described previously [4 (link)]. Briefly, 7 μg of EEEV reporter RNA and 0.75 μg of Renilla reporter RNA was electroporated into RAW cells (6x10⁶ cells) using the Neon Transfection system and a 100 μl tip (1750V, 25 ms, 1 pulse). The electroporated cells was equally distributed in triplicate for each time point. At each time point, cells were washed with PBS and lysed with 1X Passive lysis buffer (PLB). The Dual Luciferase Assay (Promega) was performed according to manufactures guidelines using 25 μl sample and 25 μl of each reagent. Relative light units (RLUs) were measure using an injection system and an Orion microplate luminometer (Berthold). Firefly RLUs were normalized to Renilla RLUs at each time point.

To determine the effect of PYHIN proteins, Sp1 and p65 on the activity of different viral promoters, 22,000 HEK293T cells/well were seeded in poly-L-lysine-coated 96-well plates and transfected in triplicates using the calcium-phosphate transfection method. Cells were cotransfected with firefly luciferase reporter constructs under the control of the HIV-1 LTR (0.3 μg) or the CMV IE promoter (2 μg) and either expression constructs for PYHIN proteins (50 ng), increasing doses of an expression construct for Sp1 or p65, or a vector control. When indicated, an expression construct for HIV-1 NL4-3 Tat (500 pg) was cotransfected to activate the LTR promoter. 40 h post-transfection, cells were lysed and firefly luciferase activity was determined using the Luciferase Assay Kit (Promega) according to the manufacturer’s instructions with an Orion microplate luminometer (Berthold).