Monitoring Stage-Specific Gene Expression in Vaccinia Virus Using Fluorescent Reporters

Abstract

Source: Rozelle, D. K., et al. Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression. J. Vis. Exp. (2014)

This video demonstrates a method to monitor vaccinia virus replication using fluorescent reporters regulated by stage-specific promoters. Sequential expression of green, red, and blue fluorescent proteins marks the early, intermediate, and late phases of infection, enabling high-throughput analysis of stage-specific viral gene expression.

Protocol

1. Plate Cells

1. Dissociate HeLa cells from plate and dilute in growth media (Dulbecco's Modified Eagle Medium [DMEM], 2 mM L-glut, 10% fetal bovine serum [FBS]) to approximately 2.0 x 10⁵ cells/ml. Dispense 100 μl/well in a black-walled, clear flat-bottom 96-well plate (20,000 cells/well).
2. Incubate cells for 24 hr until confluent in a 37 °C incubator + 5% CO₂.

2. Infect cells

1. Dilute viruses and infect cells.
   1. Thaw TrpV (Triple virus; Early Venus, Intermediate mCherry, Late TagBFP) and PLV (Promoter-less Venus) fluorescent virus and disaggregate using sonication for 5 minutes. Alternatively, crude virus stocks can be mixed 1:1 with 0.25 mg/ml trypsin in infection media and incubated at 37 °C for 30 min.
   2. Dilute crude virus stocks in 37 °C infection media (DMEM, 2 mM L-glut, 2% FBS). For high MOI infections (10 PFU/cell), dilute virus stock to 1.0 x 10⁷ PFU/ml assuming 5 x 10⁴ cells/well and an inoculum volume of 50 μl. Plan on infecting 3 replicate wells for each treatment with TrpV and another 3 replicate wells for the same treatment with PLV to account for background fluorescence specific to each treatment.
   3. Infect wells by adding 50 μl/well diluted virus in infection media. This is defined as time = 0 hr post infection (0 hpi).
2. Incubate 6-24 hr in a 37 °C incubator + 5% CO₂.

3. Fix cells

1. Fix cells by adding 100 μl 8% paraformaldehyde to infection media already in each well. Incubate at room temperature for 15 min protected from light.
   NOTE: It is recommended to add 2x Paraformaldehyde (8%) directly to infection media to prevent aerosolization of infectious vaccinia which can occur if high-titer media is inverted directly into the waste dish prior to fixation.
2. Remove the fixative by inverting the plate into the waste dish.
3. Add 100 μl of room temperature phosphate-buffered saline.
4. Seal plate with optically clear adhesive film.
   NOTE: Plates can be stored at 4 °C if not read immediately. Be sure to return sealed plates to room temperature before reading to prevent condensation from distorting spectrophotometer measurements.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	11995-065
Fetal Bovine Serum - Optima, Heat Inactivated (FBS)	Atlanta Biologicals	S12450H
200 mM L-Glutamine, (L-glut)	Gibco	25030-081
96 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates	Corning	3603
Phosphate-Buffered Saline (PBS)	Gibco	10010-023
TempPlate RT optically clear film	USA Scientific	2978-2700
Opti-MEM Reduced Serum Medium	Gibco	31985-070