A Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells

Overview

This video demonstrates an assay to study the translation regulation in poxvirus-infected cells. Uninfected and virus-infected cells were co-transfected with mRNAs encoding for firefly luciferase (Fluc) and Renilla luciferase (Rluc), with the Fluc mRNA containing a 5' Poly(A) leader sequence. Upon adding bioluminescent substrates on lysed cells, measure the signal from both the luciferases to identify the translational advantage of the Fluc mRNA in virus-infected cells, conferred by the leader sequence.

Protocol

1. Transfect mRNA to Cells

1. Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent the next day) and incubate overnight in an incubator at 37 °C with 5% CO₂.
2. Infect HeLa cells with vaccinia virus (VACV) at a Multiplicity of Infection (MOI) of 5 or keep uninfected HeLa cells for comparison.
   NOTE: MOI is the number of infectious viral particles per cell.
3. MOI of X= {[(Number of cells * X) / Virus Titer] * 1000} µl of virus per 1 mL medium.
4. After desired h post-infection (hpi) (in this experiment at 10-12 hpi), transfect mRNA (500 ng of total mRNA per well of 24-well plates) using a cationic lipid transfection reagent as shown in Figure 1C.
   1. For one well of a 24-well plate, mix 480 ng of 12A sequence bearing firefly luciferase (12A-Fluc) mRNA and 20 ng of Kozak sequence bearing Renilla luciferase (Kozak-Rluc) mRNA in one microcentrifuge tube. In another microcentrifuge tube add 1.1 µL of cationic lipid transfection reagent.
   2. Add 55 µL of reduced serum medium in both tubes. Mix and incubate at room temperature for 5 min.
   3. After 5 min of incubation, add 55 µL cationic lipid transfection reagent containing reduced serum medium in mRNA mRNA-containing tube.
   4. Mix gently but thoroughly, and incubate at room temperature for 15 min.
   5. During the incubation, remove the cell culture medium and add 400 µL of reduced serum medium per well of 24-well plates.
   6. After incubation, add 100 µL of the mixture dropwise and evenly to one well of 24-well plates.

2. Measure Luciferase Activities

1. Five hours post-co-transfection of 12A-Fluc and Kozak-Rluc mRNA, measure luciferase activity using a luciferase assay system capable of performing two reporter assays (e.g., Dual-Luciferase Reporter assay kit).
2. Remove the reduced serum medium and lyse the cells by adding 150 µL 1x lysis buffer, a component of the luciferase assay kit.
3. After 10 min incubation at room temperature, collect the lysate by scrapping the cells and transfer them to a microcentrifuge tube.
4. Centrifuge the lysate at 12,000 x g for 10 min at 4 °C to pellet cell debris.
5. Add 30 µL of supernatant in an opaque-walled 96-well white assay plate with a solid bottom.
6. Measure the dual luminescence using the luciferase assay kit and a multimode plate reader luminometer.
7. Perform the measurement using the kinetics function (on a per-well basis) using the settings described in Table 1.
   NOTE: The reading can also be taken using a manual luminometer. Add an equal volume of lysate and substrate for Fluc in a cuvette. Wait for 2 s and measure for 10 s using a luminometer. Following the Fluc measurement, quickly take out the cuvette from the luminometer and add an equal volume of the substrate for Rluc manually. Again, wait for 2 s and measure for 10 seconds using a luminometer.
8. Export the luminescence reading data into a desirable file format.
9. Determine the relative translation rate from 12A-Fluc mRNA in uninfected and VACV-infected HeLa cells by dividing the Fluc value by the internal control Rluc value.
   NOTE: Figure 2 shows the step-by-step analysis of raw data to get relative Fluc activity.

Table 1: Luciferase Measurement Settings – the steps for luciferase measurement with the recommended volume or time.

Steps	Volume/Time
Inject Luciferase Assay Substrate (Fluc):	30 µL
Wait / Incubation time:	2 s
Luminescence Measurement (Fluc):	10 s
Stop & Glo Substrate (Rluc):	30 µL
Wait / Incubation time:	2 s
Luminescence Measurement (Rluc):	10 s

Representative Results

Figure 1: mRNA synthesis and transfection. (A) Schematic of in vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5'-UTR of interest and the T7 promoter is used as the template. The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5' to 3' direction. Once mRNA is 25-30 nt long, m7G cap is added using an anti-reverse cap analog, ARCA. (B) RNA bands from in vitro transcription were detected using 1.5% agarose TBE gel electrophoresis. (C) Schematic demonstrating the transfection of reporter mRNA into cells. Medium containing either the reporter mRNA or cationic lipid transfection reagent in separate tubes is allowed to equilibrate at room temperature for 5 min. The solutions are then mixed followed by incubation at room temperature for 15 min after which the RNA/transfection reagent mixture is added to cells in culture plates.

Figure 2: Analysis of raw data – steps for analyzing raw data to get normalized data.

Materials

Name	Company	Catalog Number	Comments
Corning 96 Well Half-Area white flat bottom polystyrene microplate	Corning	3693	Opaque walled 96 well white plate with solid bottom
Dual-Luciferase Reporter Assay System	Promega	E1960	Dual-Luciferase Assay Kit (DLAK)
GloMax Navigator Microplate Luminometer	Promega	GM2010	Referred as multimode plate reader luminometer
Lipofectamine 2000	Thermo Fisher Scientific	11668019	Cationic lipid transfection reagent
Opti-MEM	Thermo Fisher Scientific	31985070	Reduced serum media