Generation of GFP-Tagged Recombinant Respiratory Syncytial Virus Using a T7 Expression System

Abstract

Source: Bouillier, C. et. al., Amplification, and Titration of Recombinant Respiratory Syncytial Viruses. J. Vis. Exp. (2019)

This video demonstrates the generation of a green fluorescent protein (GFP)-expressing recombinant respiratory syncytial virus using a T7-based reverse genetics system. The co-transfection of plasmids encoding viral proteins and a GFP-tagged genome leads to protein expression and initiates virus rescue.

Protocol

1. Rescue and First Passage of Recombinant Virus

NOTE: Perform all the following steps in a sterile environment, using a class II safety cabinet.

1. The day before transfection, make a suspension of the BSRT7/5 cell line at 5 x 10⁵ cells/mL in complete medium. Distribute 2 mL of cell suspension per well in a 6-well plate. Prepare one well per virus that is going to be rescued and one additional well for a negative control. Incubate the plate at 37 °C and 5% CO₂. Check that the cells are at a 80%–90% confluence the next day.
2. Unfreeze the reverse genetics respiratory syncytial virus (RSV) vectors p-RSV-GFP and p-RSV-M2-1-GFP, as well as p-N, p-P, p-L, and p-M2-1. Mix, for each virus to rescue, 1 µg of p-N and p-P, 0.5 µg of p-L, 0.25 µg of p-M2-1, and 1.25 µg of p-RSV (GFP or M2-1 GFP) in a tube.
   NOTE: Different expression vectors for viral proteins of the polymerase complex (N, P, L, and M2-1) may be used; however, the ratio between the proteins has to be maintained. Perform the negative control by replacing the p-RSV vector with an empty vector.
3. Proceed to transfection (see Table of Materials).
   1. Add 250 µL of reduced serum medium to the mixed vectors. In another tube, dilute 10 µL of the transfection reagent in 250 µL of reduced serum medium. Gently vortex both tubes and wait for 5 min. Mix the contents of both tubes and wait for 20 min at room temperature.
   2. Rinse the BSRT7/5 cells with 1 mL of reduced serum medium and distribute 1.5 mL of Minimal essential medium (MEM) with 10% fetal calf serum (FCS) without antibiotics per well. If necessary, incubate at 37 °C and 5% CO₂ until the incubation described in step 1.3.1 is completed.
   3. Add 500 µL of the transfection mix prepared in step 1.3.1 to a well when the 20 min incubation time is over. Place the cells in the incubator at 37 °C and 5% CO₂ for 3 days. Do not change the culture medium of the cells during the transfection.
4. Observe green fluorescent protein (GFP) fluorescence (excitation at 488 nm and emission at 515–535 nm) under an inverted fluorescence microscope at 20x magnification 1x per day to monitor the rescue efficiency, using the GFP filter.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSRT7/5		not commercially available	See reference 22. Buchholz et al. 1999
Fluorescence microscope for observations	Olympus	IX73 Olympus microscope
Fluorescence microscope for videomicroscopy	Olympus	ScanR Olympus microscope
HEp-2	ATCC	ATCC CCL-23
HEPES ≥99.5%	Sigma	H3375
L-Glutamine (200 mM)	ThermoFisher Scientific	25030024
Lipofactamine 2000 reagent	ThermoFisher Scientific	11668019	Protocol in step 2.3. is optimized for this reagent.
MEM (10x), no glutamine	ThermoFisher Scientific	11430030
MEM, GlutaMAX supplement	ThermoFisher Scientific	41090-028
MgSO4 ReagentPlus, ≥99.5%	Sigma	M7506
Opti-MEM I reduced serum medium	ThermoFisher Scientific	51985-026
Plasmids		not commercially available	See reference 21. Rameix-Welti et al. 2014