Article Figures & Data
Figures
- FIG. 1.
RSV replication in normal NHBE, SAEC, and the A549 cell line following infection with an input MOI of 2. (A) Kinetics of accumulation of cell-associated RSV and free virus released into the medium. The data represent the average titers from three wells of six-well plates per time point for each type of cell. (B) Photomicrographs illustrating cytopathological changes in NHBE, SAEC, and A549 cells at various time points following RSV infection at an MOI of 2. The 0-h time point represents the cells immediately before the infection. Mock-infected cells incubated and photographed in parallel at all time points looked similar to that at 0 h and are not shown.
- FIG. 2.
Kinetics of apoptosis induced by RSV. (A) Quantitation of annexin V-positive cells by flow cytometry in the primary NHBE and SAEC cells and A549 cells. The percentages of annexin V-positive cells infected with RSV, UV-RSV, or influenza virus or mock infected are shown as the mean of two samples per group for the primary cells and four samples per group for A549 cells. (B) TUNEL assay for apoptosis of RSV-infected or mock-infected A549 cells. The results are expressed as the ratio of the median fluorescence for RSV-infected cells to that for mock-infected cells (fluorescent infected cells/fluorescent uninfected cells). The experiment was performed three times, and representative data are shown. A positive control for DNA fragmentation obtained by treatment of uninfected cells with DNase I (not shown) gave results similar to the RSV-infected culture at 72 h; a negative control using unlabeled RSV-infected cells (not shown) gave results similar to the mock-infected control at 24 h. (C) Fluorescence microscopy showing chromatin condensation (arrow) in A549 cells at 48 h postinfection. Only a minority (<10%) of the RSV-infected culture showed chromatin condensation; similar cells were not observed in the uninfected control.
- FIG. 3.
Induction and activation of intracellular caspases in response to RSV infection. (A) Flow cytometry analysis of activated caspases in RSV-infected A549 cells harvested at the indicated times postinfection. The cells were labeled with FITC-VAD-FMK, and the percentage of positive cells is indicated in each panel. The experiment was performed three times, and representative data are shown. (B) Kinetics of accumulation of mRNA for caspases 8, 10, 3, 6, and 7 in NHBE, SAEC, and A549 and HEp-2 cells (caspases 8 and 10 are initiator caspases; caspases 3, 6, and 7 are effector caspases). RPA was performed, and the level of each caspase mRNA is expressed as a percentage of the GAPDH housekeeping mRNA. Note the differences in the scale of the y axis. (C) Kinetics of enzymatic activity for caspases 3, 6, 8, and 9 in RSV-infected A549 cells. Each experiment was performed twice, and representative data are shown.
- FIG.4.
RSV induces expression of pro- and antiapoptotic members of the Bcl-2-family. (A and B) Kinetics of accumulation of mRNA for (A) proapoptotic proteins Bid, Bax, and Bak and (B) antiapoptotic proteins Mcl-1, Bcl-W, and Bcl-xL in NHBE, SAEC, A549, and HEp-2 cells that were mock-infected or infected with RSV or UV-inactivated RSV. RPAs were performed and the level of each Bcl-2 family member mRNA is shown relative to that of GADPH mRNA measured in the same gel lane. (C) Expression of Mcl-1 protein in RSV-infected A549 cells is shown at various times postinfection by intracellular immunostaining and flow cytometry. The median fluorescence ± standard error was derived from three samples in each group. (D) Western blot analysis of Mcl-1 protein in RSV-infected or mock-infected A549 cells harvested 12 or 24 h postinfection; Mcl-1 is 37 kDa (34). (E) Immunohistochemical staining of Mcl-1 in RSV-infected A549 cells. A549 cells were infected with RSV or mock infected, fixed 24 h postinfection, and stained with anti-Mcl-1 antibody followed by an FITC-conjugated secondary antibody.
- FIG. 5.
Induction of TRAIL mRNA and protein in response to RSV infection. (A) Production of TRAIL mRNA in RSV-, UV-RSV-, or mock-infected A549 cells, NHBE, and SAEC. Total intracellular RNA was harvested at the indicated times postinfection, and the amount of TRAIL mRNA measured by an RPA is shown as a percentage of the GAPDH mRNA measured in the same gel lane. (B) Expression of transmembrane TRAIL protein at the surface of RSV- or mock-infected A549 cells measured by flow cytometry at the indicated times postinfection. The percentage of TRAIL-positive cells is expressed as the mean ± standard error based on three samples per group. Results obtained with an isotype control antibody were similar to those obtained with mock-infected control cells (not shown).
- FIG. 6.
Induction of the DR4 and DR5 TRAIL receptors in RSV-infected NHBE, SAEC, and A549 and HEp-2 cells. (A) Kinetics of DR4 and DR5 mRNA accumulation in the four different cell cultures. Intracellular RNA was isolated at the indicated times and the amount of DR4 and DR5 mRNA was determined by an RPA and is represented here as a percentage of the GAPDH mRNA. (B) Expression of DR4 and DR5 protein at the surface of RSV-infected A549 cells. The numbers in each box are the ratio of the median fluorescence for RSV-infected cells to that for mock-infected cells (fluorescence infected cells/fluorescence uninfected cells), based on three samples per group. The isotype control consisted of cells processed with a normal goat IgG and was similar to mock-infected cells (not shown).
- FIG.7.
Infection with RSV sensitizes cells to TRAIL-induced apoptosis. RSV-infected or mock-infected A549 cells were treated for 5 h, beginning 24 h postinfection, with added TRAIL (500 ng/ml) in the presence of TRAIL-specific antibody or were mock treated. The cells were photographed to visualize TRAIL-induced apoptosis (panels at left) and collected and analyzed for apoptosis by annexin V staining (panels at right); the percentage of annexin V-positive-cells (bar) is indicated for each histogram. The experiment was performed four times, and representative data are shown.
- FIG. 8.
Kinetics of acquisition of sensitivity to TRAIL-induced apoptosis during RSV infection. RSV-infected A549 cells were infected, and replicate cultures were treated with TRAIL (250 ng/ml) in the presence of TRAIL-specific antibody for 5 h beginning at 6, 11, 16, 19, 22, or 25 h postinfection. A control culture (M) was mock infected and processed in parallel with the 6-h sample. The cells were collected and stained with annexin V, and the percentage of apoptotic cells was determined.
