Renilla luciferase assay reagent

HEK-293T (1–2×10⁷) cells were infected with WSN or P908/WSN recombinant viruses at a m.o.i. of 3. Six hours post-infection, cells were lysed in 0.5 ml of lysis buffer (20 mM MOPS-KOH pH 7.4, 120 mM of KCl, 0.5% Igepal), supplemented with Complete Protease Inhibitor Mixture (Roche). Cell lysates were processed and incubated wih StrepTactin beads (StrepTactin Sepharose High Performance, GE Healthcare) as described in [51] . After three washes with 1 ml of lysis buffer, protein complexes were eluted from StrepTactin beads with desthiobiotin (IBA). Purification samples were either diluted in Laemmli sample buffer and analyzed by western-blot, or diluted in Renilla lysis buffer (Promega) and submitted to Gaussia princeps luciferase enzymatic activity measurement, using the Renilla luciferase assay reagent (Promega) and a Berthold Centro XS luminometer.

The antiviral activity of the (p-BthTX-I)₂K peptide against CHIKV-NLuc was determined via the measurement of the activity of the virus-encoded NLuc reporter. After incubation, the supernatant was removed, the cells were washed with PBS (phosphate-buffered saline) solution, and 30 µL of Renilla luciferase assay lysis buffer (Promega, Madison, WI, USA) was immediately added to each well. After 30 min, the plates containing the cell lysates were placed in a plate reader (FLUOstar Omega/BMG LABTECH, Ortenberg, Germany), where 50 μL of the substrate for Renilla uciferase (Renilla Luciferase assay reagent, Promega, Madison, WI, USA) was automatically injected into the wells. The light intensity was read, and the values obtained were expressed as a percentage of expression compared with that of the vehicle control (VC) (sterile water).

Tissue homogenates were centrifuged at 12,000 rpm for 5 minutes. 40 μL of cleared homogenates was used to carry out the Renilla luciferase assay with 50 μL Renilla luciferase assay reagent (Promega, Madison, WI) and light intensity was detected by a GloMax® 96 Microplate Luminometer (Promega). Four replicates of each tissue sample from each mouse were averaged. Due to differences in homogenates, background levels of luciferase varied.

Vero E6 cells (10⁵ cells/well) seeded in 24-well plates were infected with SARS-CoV-2 at an MOI = 1 for 1 h and then transfected in duplicate with in vitro synthesized RLuc, GI.616-EMC/RLuc, and GI.616-TRS/RLuc RNA. The supernatant was collected at 24 h post-infection, clarified, and fifty percent of the virus was used in serial infections of Vero E6 cells. At 24 h post-infection of each passage, growth media was removed, cells were washed with PBS, and lysed with 100 μL renilla luciferase assay lysis buffer (Promega, #E2810) for 15 min at room temperature. The lysate was collected and stored at −80 °C until further use. For luciferase assays, 20 μL from each passage was aliquoted into white 96-well microplates (Greiner bio-one) in duplicates for Renilla luciferase activity measurements. One hundred microliters of renilla luciferase assay reagent (Promega, #E2810) was added to each well, and luciferase activity was measured immediately using a Synergy HTX plate reader (Biotek; Winooski, VT, USA). In addition to the virus supernatant used in serial infections, the remaining virus was collected for titering. Additionally, total cellular RNA from each passage of cells was also extracted and stored at −80 °C for future use.

Measurement of the activity of the virus-encoded NanoLuciferase (NLuc) reporter was used to evaluate the antiviral activity of the peptides against CHIKV-NLuc at the end of every assay with the peptides. Cells were infected with CHIKV-NLuc, and the supernatant was aspirated 16 h post-infection (h.p.i.), and each well of the plate was washed with PBS solution. Then, 30 µL of Renilla Luciferase Assay Lysis Buffer (Promega, Madison, WI, USA) was added to the cells. After 30 min, 50 μL of substrate for Renilla Luciferase (Renilla Luciferase assay reagent, Promega, Madison, WI, USA) was automatically injected in the wells into the plate reader (FLUOstar Omega/BMG LABTECH, Ortenberg, Germany), and the light intensity reading was performed. The values obtained are shown as the percentage of protein NLuc activity in the control (samples treated with sterile water).