rM51R-M virus induces caspase-8-like and caspase-9-like activities. Fluorometric caspase assays were performed using substrates for caspase-8 (A) and caspase-9 (B). L929 cells were left untreated (Mock), infected with rwt virus or rM51R-M virus, treated with 50 nM TRAIL (A), or treated with 1 μM SSP (B). At 8 (black bars), 16 (hatched bars), and 24 (white bars) h postinfection, cells were lysed and caspase activities were measured with fluorogenic substrates. Caspase activities are expressed in arbitrary fluorescence units per milligram of total protein and are normalized to the maximum values expressed by the positive controls, TRAIL and SSP, respectively. The data represent the averages ± SEM from three experiments. hpi, hours postinfection; *, P is <0.05 relative to mock-infected samples; **, P is <0.001 relative to rM51R-M virus-infected samples at 16 h postinfection.

Inhibition of caspase-9 activity does not delay apoptosis induction by the rM51R-M virus. L929 cells that overexpress Bcl-2- (L929-Bcl-2) or an empty vector control (L929-Bcl-2-EV) were generated. (A) Cell lysates were generated and analyzed via Western blotting as described in the legend to Fig. 3, using an antibody against Bcl-2. (B and C) L929-Bcl-2 and L929-Bcl-2-EV cells were mock infected (M) or infected with rM51R-M virus. At the indicated times postinfection, cell lysates were obtained and analyzed via Western blotting as described above, utilizing an antibody against caspase-9. A representative gel is depicted in panel B. Panel C shows quantitation of procaspase-9 expression at 4 (black bars), 8 (hatched bars), and 16 (white bars) h postinfection. These values are normalized to actin protein expression and are shown as percentages of mock-infected samples. The data represent the averages ± SEM from three experiments. hpi, hours postinfection; *, P is <0.01 relative to mock-infected L929-Bcl-2-EV cells. (D) L929-Bcl-2 cells (open symbols) and L929-Bcl-2-EV cells (filled symbols) were infected with the rM51R-M virus (squares) or treated with 1 μM SSP (circles). Cells entering apoptosis were monitored by time-lapse video microscopy as described in the legend to Fig. 1. The data represent the averages ± SEM from three experiments.

Inhibition of caspase-8 activity delays apoptosis induction by rM51R-M virus. (A and B) L929 cells were left untreated or pretreated for 1 h with a synthetic caspase-8 inhibitor, Z-IETD-CHO (10 μM), and then mock infected (M) or infected with rM51R-M virus. At the indicated times posttreatment, cell lysates were prepared and analyzed via Western blotting as described in the legend to Fig. 3, using an antibody against caspase-8. A representative gel is depicted in panel A. Panel B shows the quantitation of procaspase-8 expression at 4 (black bars), 8 (hatched bars), and 16 (white bars) h postinfection. These values are normalized to actin protein expression and are presented as percentages of mock-infected samples. The data represent the averages ± SEM from three experiments. *, P is <0.05 relative to untreated, mock-infected L929 cells. (C) L929 cells were left untreated (filled symbols) or pretreated with 10 μM Z-IETD-CHO (open symbols) and then infected with rM51R-M virus (squares) or treated with 50 nM TRAIL (circles). Cells entering apoptosis were monitored by time-lapse video microscopy as described in the legend to Fig. 1. The data represent the averages ± SEM from three experiments.

VSV induces two distinct mechanisms of apoptosis. In rwt virus-infected cells (left), wt M protein inhibits host gene expression. This cellular stress results in the activation of the mitochondrial apoptotic pathway, which involves the activation of caspase-9 and, subsequently, caspase-3. Caspase-8 is activated by cross talk (dashed lines) in this pathway. rM51R-M virus infection (right) induces proapoptotic gene expression. Proapoptotic gene products then activate caspase-8 and, subsequently, caspase-3. It is possible that caspase-8 is activated directly or following the activation of death receptors. Caspase-9 is activated by cross talk in this pathway. Independent of the activation of caspase-8, rM51R-M virus infection induces ER stress, resulting in the activation of caspase-12.

VSV expressing mutant M protein induces apoptosis in L929 cells more rapidly than VSV expressing wt M protein. (A) For the time-lapse microscopy analysis, L929 cells were infected with rwt virus (filled circles), rM51R-M virus (filled squares), or rM51R-M virus in the presence of actinomycin D (open squares) or were treated with actinomycin D alone (open circles). The cumulative percentage of cells entering apoptosis was determined as the number of cells that underwent membrane blebbing followed by other morphological changes consistent with apoptosis and is plotted as a function of time postinfection. The data represent the averages ± standard errors of the means (SEM) from three experiments. (B) Caspase-3 activity was assayed in L929 cells that were mock infected or infected with rwt virus or rM51R-M virus for 16 (black bars) or 24 (white bars) h postinfection. Caspase-3 activities in cell lysates were measured with a fluorogenic substrate and are expressed in arbitrary fluorescence units per milligram of total protein, and these data are normalized to the maximum value, expressed by the rM51R-M virus at 24 h postinfection. The data represent the averages ± SEM from three experiments. (C) Mitochondrial transmembrane potential was measured in mock-infected, rwt virus-infected, or rM51R-M virus-infected L929 cells. At 16 (black bars) and 24 (white bars) h postinfection, cells were labeled with DiOC₆ and analyzed via flow cytometry. The data are expressed as the mean fluorescence intensity (MFI) of each sample as a percentage of the mock-infected cells. The data represent the averages ± standard deviations for two experiments. (B and C) hpi, hours postinfection.

rM51R-M virus induces cleavage of procaspases. L929 cells were mock infected (M) or infected with rwt virus or rM51R-M virus. At the indicated times postinfection, cell lysates were generated and separated via SDS-PAGE on a 12% polyacrylamide gel, transferred to polyvinylidene difluoride, and analyzed via Western blotting with antibodies for caspase-8 (A and B), caspase-9 (C and D), and caspase-12 (E and F). Representative gels are depicted in panels A, C, and E. Panels B, D, and F show quantitation of procaspase protein expression at 4 (black bars), 8 (hatched bars), and 16 (white bars) h postinfection. These data are normalized to actin protein expression and are shown as percentages of mock-infected samples. The data represent the averages ± SEM from four experiments. hpi, hours postinfection *, P is <0.05 relative to mock-infected samples.

Inhibition of caspase-12 activity does not delay apoptosis induction by rM51R-M virus. L929 cells were generated that overexpress MAGE-3 (L929-MAGE-3) or an empty vector control (L929-MAGE-3-EV). (A) Cell lysates were generated and analyzed via Western blotting as described in the legend to Fig. 3, using an antibody against members of the human MAGE-A family, which includes MAGE-3. (B and C) L929-MAGE-3 and L929-MAGE-3-EV cells were mock infected (M) or infected with the rM51R-M virus. At the indicated times postinfection, cell lysates were obtained and analyzed via Western blotting utilizing an antibody against caspase-12. (B) A representative gel. (C) Quantitation of procaspase-12 expression at 4 (black bars), 8 (hatched bars), and 16 (white bars) h postinfection. These values are normalized to actin protein expression and are presented as percentages of mock-infected samples. The data represent the averages ± SEM from four experiments. hpi, hours postinfection; *, P is <0.05 relative to L929-MAGE-3-EV cells at 4 h postinfection. (D) L929-MAGE-3 (circles) and L929-MAGE-3-EV (squares) cells were infected with rM51R-M virus. Cells entering apoptosis were monitored by time-lapse video microscopy as described in the legend to Fig. 1. The data represent the averages ± SEM from three experiments.

Inhibition of caspase-8 activity inhibits activation of caspase-9 but not caspase-12. L929 cells were left untreated or pretreated for 1 h with 10 μM Z-IETD-CHO and then mock infected (M) or infected with the rM51R-M virus. At the indicated times postinfection, cell lysates were prepared and analyzed via Western blotting as described in the legend to Fig. 3, utilizing antibodies against caspase-9 (A and B) and caspase-12 (C and D). Representative gels are depicted in panels A and C. Panels B and D show quantitation of procaspase protein expression at 4 (black bars), 8 (hatched bars), and 16 (white bars) h postinfection. These values are normalized to actin protein expression and are presented as percentages of mock-infected samples. The data represent the averages ± SEM from four experiments. (B and D) hpi, hours postinfection; *, P is <0.05 relative to untreated, mock-infected L929 cells; **, P is <0.05 relative to Z-IETD-CHO- pretreated L929 cells at 8 h postinfection.