Assessing Viral Infectivity Using a β-Galactosidase-Based Assay

Resumen

Source: Tripathi, A., et al. Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication. J. Vis. Exp. (2024)

This video demonstrates the assessment of viral infectivity using reporter epithelial cells engineered with a β-galactosidase gene under a viral protein-sensitive promoter. Following infection with different viral dilutions, the infected host cells express β-galactosidase, which is detected using a chromogenic substrate. The resulting stained cells are then counted to quantify infective virion particles across varying virus concentrations.

Protocolo

1. Quantitation of virus concentration and virion infectivity
   1. Count and seed 0.1 x 10⁶ TZM-bl cells in a 24-well plate using 500 µL of complete Dulbecco's Modified Eagle Medium (DMEM) for each well and keep them in a cell culture incubator for 10-12 h.
   2. Make sure the cells are 50%-60% confluent before beginning with infection for virus quantitation. Change the existing media with 250 µL of fresh complete DMEM to each well.
      NOTE: Keep a positive control well to rule out any experimental fault.
   3. Calculate the amount of stock virus needed for infections of 1 ng, 0.1 ng, and 0.01 ng in duplicates. Add the appropriate amount of virus to the wells and keep the plate in the incubator at 37 °C for 4 h.
   4. Wash the cells twice with 500 µL of incomplete DMEM and then add 500 µL of complete DMEM.

   5. Proceed with the β-gal staining procedure after 36 h of media change.

      1. Discard the existing media and wash the cells twice with 1 mL of Phosphate-Buffered Saline (PBS). Fix the cells by adding 500 µL of the fixing solution (0.25% Glutaraldehyde in PBS) to each well and incubate at room temperature (RT) inside the biosafety cabinet for 5-7 min.
      2. Prepare the staining solution as given in Table 1. Keep it away from light, as X-gal is light-sensitive.
      3. Wash the cells once with 1 mL of PBS. Overlay the cells with 500 µL of freshly prepared staining solution and incubate at 37 °C in the dark for 2-18 h.
      4. To stop the reaction afterward, rinse the cells with 500 µL of 3% dimethyl sulfoxide (DMSO) in PBS twice and then keep the cells in 500 µL of fresh PBS.
      5. Count the blue infected cells (as shown in Figure 1A) under the microscope at 10x magnification for 5 random fields. Take an average count of the 5 fields and multiply it by the field factor and dilution factor. This quantifies the infective virion particles in the virus stock.
         NOTE: For a 24-well plate, the field factor is 75.

      Table 1: List of components required for β-gal staining in TZM-bl cells.

      Components	Preparation	Required volume
      PBS	Prepare 1x PBS from a stock of 10x PBS	14 mL
      Potassium Ferri-ferrocyanide	Dissolve 0.82 g of Potassium Ferricyanide and 1.06 g of Potassium Ferrocyanide in 25 mL of 1x PBS	0.75 mL
      Magnesium chloride	1 M in 1 mL of 1x PBS	15 µL
      X-gal	Dissolve 30 mg in 0.6 mL of N, N-dimethyl formamide (in dark)	0.3 mL
      		Total = 15 mL

Resultados

Figure 1: Quantification of virion infectivity and analysis of infection progression. (A) Quantitation of virion infectivity by TZM-bl based β-gal assay. The infectivity of the virus was determined by adding 1 ng, 0.1 ng, and 0.01 ng of the virus to the TZM-bl cells, followed by β-gal staining and counting the number of blue cells. Magnification: 10x. (B) Analysis of infection progression in 0.5 MOI HIV-1 infected CEM-GFP cells based on p24 antigen capture ELISA of the culture supernatants. CEM-GFP cells were infected with 0.5 MOI of HIV-1, and the supernatants were processed for p24 ELISA. The error bars are displayed as mean ± S.E values. Asterisks indicate the significance level, which is indicated in the figures as ns = p ≥ 0.05, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and ****=p ≤ 0.0001 as analyzed by Student's t-test.

Materiales

Lista de materiales utilizados en este artículo

Nombre	Empresa	Número de catálogo	Comentarios
DMSO	Sigma-Aldrich, USA	D2650
Fetal Bovine Serum	Invitrogen, USA	16000044
Glutaraldehyde 25%	Sigma-Aldrich, USA	G6257	Infectivity assay
HEK-293T	NCCS, India
HIV-1 infectious Molecular Clone pNL4-3	NIH, AIDS Repository, USA	114
Inverted microscope	Nikon, Japan	Model: Eclipse Ti2
Magnesium chloride	Sigma-Aldrich, USA	M8266	Infectivity assay
Penicillin/Streptomycin	Invitrogen, USA	151140122
Potassium ferricyanide	Sigma-Aldrich, USA	244023	Infectivity assay
Potassium ferrocyanide	Sigma-Aldrich, USA	P3289	Infectivity assay
UltraPure X-gal	Invitrogen, USA	15520-018	Infectivity assay