Enduren live cell substrate

A dual split-protein (DSP)-based fusion cell-cell assay was used to examine viral Env-mediated cell-cell fusion activity as described previously [31 (link)]. Briefly, a total of 1.5 × 10⁴ HEK293T cells (effector cells) were seeded on a 96-well plate and incubated overnight, and then they were transfected with a mixture of an Env-expressing plasmid and a DSP_1-7 plasmid. After 24 h, 3 × 10⁴ 293FT cells expressing CXCR4/CCR5 and DSP_8-11 (target cells) were resuspended in prewarmed culture medium that contains EnduRen live-cell substrate (Promega) at a final concentration of 17 ng/μL and then transferred to the effector cell wells at equal volumes. The mixed cells were spun down to maximize cell-cell contact, and the luciferase activity was measured as described above.

The antiviral activity of JNJ-53718678 against DENV-2 was tested against Huh7-sgDV-R2H clone 1 cells^{67 (link)}. Briefly, white 384-well microtiter plates were filled in quadruplicate using a customized robot system with serial fourfold dilutions of compound in a final volume of 10 µl culture medium followed by addition of 30 µl per well Huh7-sgDV-R2H clone 1 cells. Two days post-virus exposure, viral replication was quantified by adding 40 µl per well EnduRen™ Live Cell Substrate (Promega, Mannheim, Germany) and by measuring luminescence. The toxicity of JNJ-53718678 was tested using original Huh7 cells with an ATP read-out after 3 days similar as described for RSV.

For the fusion assay, cell lines stably expressing DSP were utilized (see above), the stable cell line expressing dual split protein DSP_8–11, were transfected with expression vectors of interest in quadruplicate. At 48 h post-transfection, 293CD4/DSP_1–7 cells (2×10⁴), a stable cell line expressing CD4 and DSP_1–7, were co-cultured with transfected 293FT/DSP_8–11 cells at 37°C in fresh medium containing membrane-permeable Enduren Live Cell Substrate (Promega). The RL activity was measured at 2hr after co-culture using GloMax-Multi Plus Detection System (Promega).
For designated experiments, the same batch of transfected 293FT/DSP8-11 cells were subjected to FACS analysis as described above. The RL activity readings were normalized to the respective MFI to compensate for the differential surface expression level of Env.
For DSP assay of cells after staining with Halo ligand, transfected cells were labeled for 15 min at 37°C with 1 µM of HaloTag TMR or Alexa Fluor 488 ligand. After labeling, cells were rinsed three times with 200 µl prewarmed DMEM/FBS and subsequently incubated at 37°C with 5% CO₂ for 30 min. After the medium was replaced with fresh warm DMEM/FBS, images were captured using a microscope, then this sample was used directly for DSP assay.

SARS-CoV-2 S protein-mediated cell–cell fusion activity was determined by a dual-split-protein (DSP)-based cell–cell fusion assay as described previously [11 (link)]. In brief, a total of 1.5 × 10⁴ 293T cells (effector cells) were seeded in a 96-well plate and 1.5×10⁵/ml 293T/ACE2 cells (target cells) were seeded in a 10-cm culture dish, and then the cells were incubated at 37°C overnight. The effector cells were cotransfected with a mixture of a SARS-CoV-2 S-expressing plasmid and a DSP_1–7 plasmid, the target cells were transfected with a DSP_8–11 plasmid, and then the cells were incubated at 37°C for 24 h. To measure the inhibitory activities of IPB02V3 and IPB24, a serially 3-fold-diluted lipopeptide was added to the effector cells and incubated for 1 h, while the target cells were resuspended at 3 × 10⁵/ml in prewarmed culture medium containing EnduRen live cell substrate (Promega) at a final concentration of 17 ng/ml and incubated for 30 min. Then, 3 × 10⁴ of target cells were transferred to the effector cells and the cell mixture was spun down to facilitate cell–cell contact. Luciferase activity was measured at different time points as described above.

In an adaptation of a published assay (46 (link)), in the first step, 1.5 μg of full-length recombinant pNLRepRuc DNA variants and 1.5 μg of plasmid SV-A-MuLV-env (murine leukemia virus [MuLV] envelope gene under the control of the simian virus 40 [SV40] promoter, obtained from the NIH AIDS Research and Reference Reagent Program; catalog number 1065) were cotransfected (calcium phosphate; Life Technologies) into 293T cells (60 to 70% confluence; 6-well plate). After an overnight incubation at 37°C and 5% CO₂, transfected cells were washed, trypsin treated, and then coseeded with untransfected 293T cells onto compound-containing 384-well plates at the cell number ratio of 1:5 to a final cell density of 9,500 cells/well. After 72 h of incubation (37°C and 5% CO₂), cell-associated Renilla luciferase activity was measured upon the addition of EnduRen live cell substrate (Promega). EC₅₀s were calculated as for the multiple-cycle assay.

In order to quantitatively measure the effect of the different chemicals on viral replication, we used a reporter IAV derived from A/WSN/33, in which the coding sequence of the hemagglutinin protein was replaced by that of Renilla luciferase to allow real-time determination of viral replication^{62 (link)}. The different kinase inhibitors were added to the A549 cells together with the reporter virus at an MOI = 1 PFU/cell. After synchronizing infection by allowing the virus to bind to target cells for 1 h at 4 °C, the inoculum was removed and fresh culture medium containing the Renilla luciferase substrate (EnduRen Live Cell Substrate, Promega) and the indicated concentrations of the different inhibitors were added to the cells. The plates were transferred to 37 °C and real-time luminescence measurements were performed at the different time-points indicated using an EnVision Multilabel Reader (Perkin Elmer).