Renilla luciferase assay

Huh-7 cells stably transfected with the CHIKV-NCT (NonCytotoxic) replicon, were used to test the effects of SAMHD1 to CHIKV replication. CHIKV replicon cells were plated in six-well plates, treated with VLPs/Vpx and incubated at 37 °C and 5% CO₂. After 24 and 48 h of incubation, Renilla Luciferase (Rluc) activity, expressed by the CHIKV replicon, was detected using the Renilla Luciferase assay (Promega, Charbonnière, France) according to the manufacturer’s instructions. The luminescence signal, proportional to the CHIKV’s RNA replication, was then measured using the Modulus microplate luminometer (Turner BioSystems, Sunnyvale, CA, USA).

To measure neutralising antibody titres, diluted plasma samples were preincubated with pseudoviruses carrying optimized sequences for the expression of spike variants 614G (B.1), Beta (B.1.351) Delta (B.1.617.2), Mu (B.1.621) and Omicron (B.1.1.529). Pseudoviral particles were generated by cotransfection of the plasmid pNL4-3ΔenvRen and expression vectors for the different viral spike variants cloned in the pcDNA3·1-SCoV2Δ19 plasmid and added at a concentration of 10 ng p24Gag per well to Vero E6 cells in 96-well plates. At 48 h post infection, viral infectivity was assessed by measuring luciferase activity (Renilla Luciferase Assay, Promega, Madison, WI, USA) using a 96-well plate luminometer LB 960 Centro XS³ (Berthold Technologies, Oak Ridge, TN, USA). The titre of neutralising antibodies was calculated as 50% inhibitory dose (neutralising titre 50, NT50), expressed as the reciprocal of four-fold serial dilution of heat-inactivated sera (range 1:32–1:131·072), resulting in a 50% reduction of pseudovirus infection compared with control. Samples below the detection threshold (1:32 serum dilution) were given 1:16 value. Non-specific neutralisation was assessed using a related pseudovirus expressing the vesicular stomatitis virus envelope.

The sequences of Ctss 3′-UTR comprising the miR-4498 binding site were synthesized and inserted into the pMIR-REPORT^TM vector (Ambion) to construct a luciferase vector. Next, miRNA mimics/miR-NC (synthesized by GenePharma, Shanghai, China) and the above-mentioned luciferase vector were co-transfected into cells. The luminescence signal was detected by GloMax^® 20/20 Luminometer (Promega) in 48 h after co-transfection in accordance with the protocol of Dual-Glo luciferase reporter assay (Promega, WI, United States). The values of the firefly luciferase assay were normalized to the Renilla luciferase assay value from the transfected phRL-null vector (Promega).

Huh-7 cells stably transfected with the CHIKV La Réunion-NCT (Non Cytotoxic) replicon^{52 (link), 53 (link)} were used to test the effect of imipramine on CHIKV RNA replication. CHIKV replicon cells were plated in six-well plates, treated with increasing concentrations of imipramine and incubated at 37 °C and 5% CO₂. After 48 h of incubation, Renilla Luciferase (Rluc) activity, expressed by the CHIKV replicon, was detected using the Renilla Luciferase assay (Promega, Charbonnière, France). The luminescence signal, proportional to the CHIKV’s RNA replication, was then measured using the Modulus microplate luminometer (Turner BioSystems, CA) and plotted to determine the antiviral activity of imipramine. Vehicle-treated cells were used as a control. A sigmoidal curve fit with variable slope was created to obtain the half maximal effective concentration (EC₅₀) value using Graph Pad Prism 5 (Graph Pad Software Inc., San Diego, CA).

Cells were seeded on 96-well plates (Greiner Bio-One) in triplicates and incubated for 16–24 h. Cells were first pre-chilled on ice for 30 min. Next, CV-B3-RLuc (MOI  =  1 TCID₅₀) was pre-bound to cells on ice for 60 min. The cells were then incubated at 37 °C in an incubator for 10 min to permit viral entry. Next, the cells were washed three times with PBS and cultured in complete culture media. At the indicated time points, total cell lysates were harvested in Renilla Luciferase Assay lysis buffer, and luciferase expression was measured using a Renilla Luciferase Assay system (E2820; Promega). Luciferase activity was measured by the addition of substrate, and luciferase readings were taken immediately using a GloMax 20/20 Luminometer with a 5-s integration time.

Cf2Th cells stably expressing human or primate CD4 and human CCR5, and 293T cells, were plated at 3 x 10⁵ and 1 x 10⁶ cells/well of a 6-well dish, respectively. The next day, 1) Cf2Th cells were transfected with 2.5 μg of DNA (1.25 μg of ½ renilla luciferase [79 (link)] and 1.25 μg of pCDNA3.1 filler DNA) using Lipofectamine 3000 (Invitrogen) and 2) 293T cells were transfected with 2.5 μg of DNA (1.25 μg of ½ renilla luciferase and 1.25 μg of the Env expression plasmid) using TransIT 293 (Mirus). Twenty-four hours post transfection, the cells were removed from the plate using an enzyme free 1X citric saline solution (10X solution, 1.35M KCl, 0.15M Sodium Citrate) diluted in PBS, counted, and resuspended to a final volume of 1x10⁵ cells/ml. 100 μl of transfected cells in quadrupilicate (1 x 10⁴ 293T and Cf2Th cells) were mixed 1:1 and incubated for 6 hours in 96-well flat bottom plates. Following incubation, fusion was assessed using a Renilla luciferase assay (Promega). Luminescence was determined using the BMG Clariostar plate reader.

Cells were harvested for luciferase activity 48 hours after transfection. Cells were lysed in 1X passive lysis buffer (Promega). To measure luciferase, LARII (Promega) was normalized by Renilla luciferase assay (Promega). All luciferase assays were performed in triplicate (n≥3).