Immunofluorescence microscopy and flow cytometry characterization of chemical induction of latent Epstein-Barr virus

Abstract

The effects of chemical induction of latent Epstein-Barr virus (EBV) with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and n-butyrate on cell viability and induction of latent EBV in Raji and X50-7 B lymphocytes, indicated by expression of the diffuse component of the EBV early antigen (EA-D), were measured by visual immunofluorescence microscopy (of both viable and nonviable cells) and fluorescence-activated cell sorter (FACS) flow cytometry (of viable cells only). Cell viability at 4 days decreased moderately for treated Raji cells (9 to 37%, compared to 55 to 69% for untreated cells) and markedly for X50-7 cells (1-32% compared to 35-44% in untreated cells). The highest EA-D levels in viable cells occurred in Raji cells treated with both TPA and n-butyrate and untreated X50-7 cells. TPA and n-butyrate acted synergistically to induce latent EBV, resulting in increased levels of EA-D production in Raji cells and cell death in X50-7 cells. Methodological differences including the ability to detect antigen in only viable cells by FACS flow cytometry accounted for the higher levels of EA-D observed by FACS analysis compared to the levels observed by immunofluorescence microscopy. FACS analysis may be more objective and reproducible than immunofluorescence microscopy for the detection of EBV induction and also permits viral protein expression to be distinguished in the subpopulation of viable cells.

FIG. 1

Observed values (circles) and estimated means and 95% confidence intervals (bars) for viability of Raji cells and X50-7 cells growing at a slow rate and a fast rate following chemical induction of latent EBV by TPA (20 ng/ml), n-butyrate (4 mM), or both compounds measured by FACS analysis at 4 days. Complete viral replication is constitutively inhibited in Raji cells (17, 35, 38), resulting in the accumulation of EA; the increased rates of death for treated cells compared to that for untreated cells is the result of the direct toxicity of TPA and n-butyrate (1, 2). Induction of EBV in X50-7 cells continues to complete viral replication with resultant cell death; increased rates of death for treated cells compared to that for untreated cells is the result of the combination of chemical toxicity plus lytic viral replication. The error bars for X50-7 cells are significantly larger because of the reversal of the results (for slow or fast growth) for TPA treatment alone compared to the results for all other groups (untreated and treated Raji and X50-7 cells).

FIG. 2

Effect of chemical induction of EBV in Raji cells and X50-7 cells (both growing at a fast rate) with TPA (20 ng/ml), n-butyrate (4 mM), and both chemicals at 4 days determined by FACS analysis with an EBV EA-D antibody. Each plot represents 20,000 viable lymphocytes (nonviable cells were excluded from FACS analysis). The percentage of cells that expressed EA-D by FACS analysis was determined separately for each experimental group at the log relative fluorescence intensity at which 1% of control cells stained only with the secondary antibody (the FITC-conjugated goat anti-mouse IgG) showed fluorescence. For Raji cells, slightly increased levels of expression of EA-D are detectable with each chemical (0.6 to 2.2%) compared to level of expression for untreated cells (0.3 to 0.6%), but the level of EA-D expression is increased 8 to 48 times when both chemicals are used compared to the level of EA-D expression after induction with either chemical alone. For X50-7 cells, the highest levels of EA-D expression detectable by FACS analysis (viable cells only) are those of untreated cells (11.3 to 22.2%) compared to the level of expression after treatment with either chemical alone (0.6 to 8.0%) or both chemicals (0.2 to 1.4%).

FIG. 3

Effect of rate of cell growth rate (slow or fast) on chemical induction of EBV in Raji cells and X50-7 cells with the combination of TPA (20 ng/ml) and n-butyrate (4 mM) at 2 and 4 days determined by FACS analysis with an EBV EA-D antibody. Each plot represents 20,000 viable lymphocytes (nonviable cells were excluded from FACS analysis). The percentage of cells that expressed EA-D by FACS analysis was determined separately for each experimental group at the log relative fluorescence intensity at which 1% of control cells stained only with the secondary antibody (the FITC-conjugated goat anti-mouse IgG) showed fluorescence. For Raji cells, the levels of EA-D are similar for both slowly growing and fast-growing cells at 2 days (2.1 and 3.4%, respectively) and at 4 days (28.5 and 18.3%, respectively; P = 0.8). For X50-7 cells, the levels of EA-D are also similar at 2 days for both slowly growing and fast-growing cells (0.2 and 0.6%, respectively) and are slightly higher at 4 days in cells growing at a fast rate than in cells growing at a slow rate (1.4 and 0.5%, respectively).

FIG. 4

Observed values (circles) and estimated means and 95% confidence intervals (bars) for EA-D expression in viable cells only following induction of latent EBV in Raji cells and X50-7 cells growing at a slow rate and a fast rate with TPA (20 ng/ml), n-butyrate (4 mM), or both compounds measured by FACS analysis at 4 days. The combination of TPA plus n-butyrate resulted in a synergistic induction of latent EBV as evidenced by the marked decrease in cell viability (Fig. 1) and the increased level of EA-D production in viable cells.