Nano glo kit

The P1 region of a full-length EV-A71²⁴ infectious clone was substituted with the gene encoding for Nano-luciferase (Promega). To generate the replicon-deficient 3D mutant, site-directed mutagenesis was performed using In-Fusion HD cloning (Clontech) to remove 53 amino acids from the C-terminus of 3D. RNA transcripts of either clone was produced using MEGAscript T7 transcription kit (Life Technologies) and products were purified using RNeasy kit (Qiagen). The transcripts were verified for integrity and concentration using agarose-gel electrophoresis before transfection. 100 ng of purified RNA transcripts of each construct were reverse-transfected into 20,000 RD cells on white 96-well plates (Corning) using Dharmafect-1 (Thermo Fisher Scientific). At 4 h post transfection, compound-containing media was substituted into each well and the samples were incubated for a further 12 h before luciferase detection using the Nano-Glo kit from Promega.

This was performed as previously described (Chen et al., 2019 (link)). The 12 strains were transformed with plasmids expressing GFP (integrated) and secreted Nluc luciferase (episomal plasmid) in two steps (Chaudhuri et al., 2018 (link)) – both GFP and Nluc were under control of a tetracycline-inducible promoter. Strains were grown overnight until OD₆₀₀ ∼ 3. Anhydrotetracycline (ATc) was added to a final concentration of 50 ng/ml to independent cultures and replicates plated in 96-well plates overnight and cultured with shaking at 37°C. The following day, 80 μL of each replicate was transferred to black, flat bottom 96-well plates (Corning #CLS3925) for GFP measurement using a Fluoroskan Ascent FL Microplate Fluorometer and Luminometer. The original culture plates were spun for 10 min to pellet bacteria and 40 μL of supernatant, which contained secreted Nluc luciferase was transferred from each well to white, flat bottom 96-well plates (Corning #CLS3922) for luminescence reading (using the Nano-Glo kit, Promega) and using the same instrument and 1000 ms integration time. Relative mistranslation rates were calculated as previously described (Chen et al., 2019 (link)) by correcting Nluc activity to GFP florescence.

Seeded 96-well plates, with 2 × 10⁴ RD cells per well, were incubated overnight at 37 °C with 5% CO₂. As described in our previous work [48 (link)], EV-A71 RNA replicons that were either replication-competent or replication-defective was used to transfect the seeded cells for 4 h at 37 °C with 5% CO₂. The transfection mixture per well was prepared as followed: EV-A71 replicon (100 ng); DharmaFECT 1 transfection reagent (0.2 µL) and serum-free DMEM (0.2 µL) for complexing before topping up with DMEM; 10% HI-FCS (80 µL). After transfection, cells were washed and treated with 0.1% DMSO, series of ZAF-47 concentrations, 1 mM GuHCl or 10 µg/mL CHX for 12 h at 37 °C with 5% CO₂. Nano-Glo kit (Promega, Madison, WI, USA) was used for luciferase detection after 12 h incubation.