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The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax.

Ilkow CS, Goping IS, Hobman TC - PLoS Pathog. (2011)

Bottom Line: The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis.Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur.Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Canada.

ABSTRACT
Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.

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Caspase 3 activation is blocked in RV infected cells.A. A549 were infected with RV (MOI = 1) for 42 hours after which they were treated with staurosporine (ST) for 6 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 20 µm. B. The percentages of cells among mock and RV infected cells treated with ST expressing active caspase 3 were determined and plotted. Student's t-Test was performed to determine statistical significance. p = ≤0.001. Percentages were determined from three independent experiments in which at least 100 cells for each experiment were scored. C. To demonstrate that ST treatment did not result in selective loss of infected cells, the percentages of RV infected cells were determined in control (RV) and ST-treated (RV + ST) samples. D. The percentage of viable cells in mock and RV infected samples were determined by Trypan blue dye exclusion at the indicated time points. Error bars indicate standard deviations calculated from three independent experiments.
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ppat-1001291-g001: Caspase 3 activation is blocked in RV infected cells.A. A549 were infected with RV (MOI = 1) for 42 hours after which they were treated with staurosporine (ST) for 6 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 20 µm. B. The percentages of cells among mock and RV infected cells treated with ST expressing active caspase 3 were determined and plotted. Student's t-Test was performed to determine statistical significance. p = ≤0.001. Percentages were determined from three independent experiments in which at least 100 cells for each experiment were scored. C. To demonstrate that ST treatment did not result in selective loss of infected cells, the percentages of RV infected cells were determined in control (RV) and ST-treated (RV + ST) samples. D. The percentage of viable cells in mock and RV infected samples were determined by Trypan blue dye exclusion at the indicated time points. Error bars indicate standard deviations calculated from three independent experiments.

Mentions: We reasoned that in order for RV replication and virion secretion to increase through 48 hours and beyond, programmed cell death must be inhibited during this period. Accordingly, we compared the levels of apoptosis in RV and mock-infected A549 cells by indirect immunofluorescence using an antibody specific for activated caspase 3. Interestingly, less than 5% of infected cells exhibited signs of apoptosis 48 hours post-infection (Figure 1A and B). Moreover, when challenged with the kinase inhibitor staurosporine, a potent inducer of apoptosis [18], RV infected cells were significantly more resistant to apoptosis than mock infected cells. Specifically, the percentage of caspase 3-positive cells was almost three fold lower in the infected samples. This was not due to detachment of infected cells as data in Figure 1C show that treatment with staurosporine did not cause significant loss of infected cells. Finally, Figure 1D shows that even after 72 hours, RV infection does not significantly affect the percentage of viable A549 cells. Together, these data indicate that RV-infected A549 cells are resistant to programmed cell death.


The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax.

Ilkow CS, Goping IS, Hobman TC - PLoS Pathog. (2011)

Caspase 3 activation is blocked in RV infected cells.A. A549 were infected with RV (MOI = 1) for 42 hours after which they were treated with staurosporine (ST) for 6 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 20 µm. B. The percentages of cells among mock and RV infected cells treated with ST expressing active caspase 3 were determined and plotted. Student's t-Test was performed to determine statistical significance. p = ≤0.001. Percentages were determined from three independent experiments in which at least 100 cells for each experiment were scored. C. To demonstrate that ST treatment did not result in selective loss of infected cells, the percentages of RV infected cells were determined in control (RV) and ST-treated (RV + ST) samples. D. The percentage of viable cells in mock and RV infected samples were determined by Trypan blue dye exclusion at the indicated time points. Error bars indicate standard deviations calculated from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g001: Caspase 3 activation is blocked in RV infected cells.A. A549 were infected with RV (MOI = 1) for 42 hours after which they were treated with staurosporine (ST) for 6 hours. Samples were then processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 20 µm. B. The percentages of cells among mock and RV infected cells treated with ST expressing active caspase 3 were determined and plotted. Student's t-Test was performed to determine statistical significance. p = ≤0.001. Percentages were determined from three independent experiments in which at least 100 cells for each experiment were scored. C. To demonstrate that ST treatment did not result in selective loss of infected cells, the percentages of RV infected cells were determined in control (RV) and ST-treated (RV + ST) samples. D. The percentage of viable cells in mock and RV infected samples were determined by Trypan blue dye exclusion at the indicated time points. Error bars indicate standard deviations calculated from three independent experiments.
Mentions: We reasoned that in order for RV replication and virion secretion to increase through 48 hours and beyond, programmed cell death must be inhibited during this period. Accordingly, we compared the levels of apoptosis in RV and mock-infected A549 cells by indirect immunofluorescence using an antibody specific for activated caspase 3. Interestingly, less than 5% of infected cells exhibited signs of apoptosis 48 hours post-infection (Figure 1A and B). Moreover, when challenged with the kinase inhibitor staurosporine, a potent inducer of apoptosis [18], RV infected cells were significantly more resistant to apoptosis than mock infected cells. Specifically, the percentage of caspase 3-positive cells was almost three fold lower in the infected samples. This was not due to detachment of infected cells as data in Figure 1C show that treatment with staurosporine did not cause significant loss of infected cells. Finally, Figure 1D shows that even after 72 hours, RV infection does not significantly affect the percentage of viable A549 cells. Together, these data indicate that RV-infected A549 cells are resistant to programmed cell death.

Bottom Line: The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis.Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur.Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Canada.

ABSTRACT
Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.

Show MeSH
Related in: MedlinePlus