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. 2003 Jan;77(1):790-8.
doi: 10.1128/jvi.77.1.790-798.2003.

Poliovirus-induced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells

Anne-Sophie Gosselin  1 , Yannick Simonin, Florence Guivel-Benhassine, Vincent Rincheval, Jean-Luc Vayssière, Bernard Mignotte, Florence Colbère-Garapin, Thérèse Couderc, Bruno Blondel
Affiliations

Poliovirus-induced apoptosis is reduced in cells expressing a mutant CD155 selected during persistent poliovirus infection in neuroblastoma cells

Anne-Sophie Gosselin et al. J Virol. 2003 Jan.

Abstract

Poliovirus (PV) can establish persistent infections in human neuroblastoma IMR-32 cells. We previously showed that during persistent infection, specific mutations were selected in the first extracellular domain of the PV receptor (CD155) of these cells (N. Pavio, T. Couderc, S. Girard, J. Y. Sgro, B. Blondel, and F. Colbère-Garapin, Virology 274:331-342, 2000). These mutations included the Ala 67 --> Thr substitution, corresponding to a previously described allelic form of the PV receptor. The mutated CD155(Thr67) and the nonmutated IMR-32 CD155 (CD155(IMR)) were expressed independently in murine LM cells lacking the CD155 gene. Following infection of the cells with PV, we analyzed the death of cells expressing these two forms of CD155. Levels of DNA fragmentation, caspase activity, and cytochrome c release were lower in LM-CD155(Thr67) cells than in LM-CD155(IMR) cells. Thus, the level of apoptosis was lower in cells expressing mutated CD155 selected during persistent PV infection in IMR-32 than in cells expressing the wild-type receptor.

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Figures

FIG. 1.
FIG. 1.
Cell death in LM-CD155IMR and LM-CD155Thr67 cells infected with PV. (A) Kinetics of the cytopathic effect in LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cells. Cells were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell and stained with hematoxylin and eosin at the times indicated. The number of cells that continued to adhere to the plate in PV-infected cultures was determined by counting cells from an area corresponding to 1,000 cells in mock-infected cultures; the percentages of adherent cells in infected cultures with respect to those in mock-infected cultures are indicated. Magnification, ×150. (B) Cell death in LM-CD155IMR and LM-CD155Thr67 cells. Two clones for each cell line were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell. At 48 h p.i., both adherent and detached cells were incubated with FDA, which is cleaved by cytoplasmic esterases only in living cells, giving rise to a fluorescent de-esterified form. The percentages of cells that were living were determined by flow cytometry. Cell death is expressed as the mean percentage of FDA-negative cells in the total population in three independent experiments. Error bars indicate the standard errors of the means.
FIG. 2.
FIG. 2.
CD155 expression and viral growth in LM-CD155IMR and LM-CD155Thr67 cells. (A) CD155 expression and percentage of PV-infected LM-CD155IMR and CD155Thr67 cells. The level of CD155 expression at the cell surface was determined for two clones for each cell line before PV infection by using flow cytometry after immunofluorescence labeling with monoclonal antibody 404.19 directed against CD155 (46). Cells positive for viral antigen (both adherent and detached) at 8 h p.i. with PV-1/Sabin at a MOI of 10 TCID50 per cell were quantified by flow cytometry after immunofluorescence labeling with monoclonal antibody C3 directed against PV capsid protein VP1 (13). Results are presented as the means of two separate experiments. Standard errors of the means are indicated, except for the percentages of cells expressing CD155, for which they are lower than 0.5. (B) Single-cycle growth curves of PV in LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cells. Cell cultures were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell. Cells and supernatants were harvested at the times indicated, and total virus yields were determined by TCID50 assay. Each point represents the mean of two separate experiments. Error bars indicate the standard errors of the means.
FIG. 3.
FIG. 3.
Kinetics of DNA fragmentation in LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cells infected with PV. Cell cultures were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell. At the times indicated, adherent cells were labeled for DNA fragmentation by TUNEL with biotin-16-dUTP and CY3-conjugated streptavidin as previously described (31). The percentage of TUNEL-positive cells was determined by counting 1,000 cells (by epifluorescence) under a Leica microscope. Results are presented as the means of two separate experiments. Error bars indicate the standard errors of the means.
FIG. 4.
FIG. 4.
Caspase activation in LM-CD155IMR and LM-CD155Thr67 cells infected with PV. (A) DNA laddering in LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cells infected with PV. Cell cultures were incubated with or without zVAD-fmk (100 μM) for 90 min and then not infected or infected by incubation with PV-1/Sabin at a MOI of 10 TCID50 per cell for 48 h in the presence or absence of zVAD-fmk (10 μM). DNA was then extracted from total cells (adherent and detached) and analyzed by electrophoresis in a 1.8% agarose gel after ethidium bromide staining. The first lane of each gel corresponds to a 100-bp DNA ladder marker (Gibco BRL). (B) Flow cytometric analysis of caspase activity in LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cells infected with PV. Cell cultures were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell. At 48 h p.i., total cells were labeled using a CaspaTag fluorescein caspase (VAD) activity kit and analyzed by flow cytometry. Histograms of the relative cell number (y axes) versus the fluorescein intensity (x axes) show two peaks, corresponding to caspase-negative cells (first peak) and caspase-positive cells (second peak). The percentages of cells with activated caspases are indicated. One experiment out of two with similar results is shown. (C) Caspase-3, caspase-9, caspase-8, and caspase-10 activity in LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cells infected with PV. Cell cultures were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell. At 24 and 48 h p.i., total cells were labeled with CaspaTag fluorescein kits for caspase-3 (DEVD) activity, caspase-9 (LEHD) activity, caspase-8 (LETD) activity, and caspase-10 (AEVD) activity and analyzed by flow cytometry. The results are expressed as the percentages of cells with activated caspases in the total population and are the means of two independent experiments. Standard errors of the means are indicated.
FIG. 5.
FIG. 5.
Cytochrome c release in LM-CD155IMR and LM-CD155Thr67 cells infected with PV. LM-CD155IMR/cl4 and LM-CD155Thr67/cl27 cell cultures were infected with PV-1/Sabin at a MOI of 10 TCID50 per cell. At the times indicated, adherent cells were stained by immunofluorescence with a specific monoclonal antibody against cytochrome c and a secondary, fluorescein-conjugated antibody. (A) Distribution of cytochrome c as observed by confocal microscopy. In mock-infected cells (left panel), cytochrome c displays a dotted pattern, consistent with its location within the mitochondria. In PV-infected cells (right), the translocation of cytochrome c into the cytosol results in a diffuse staining pattern. Magnification, ×3,200. (B) Kinetics of cytochrome c release in PV-infected cells. The percentages of cells with released cytochrome c (as shown in panel A) were determined by counting 1,000 cells by epifluorescence. Results are presented as the means of two separate experiments. Error bars indicate the standard errors of the means.
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