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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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Expression of capsid in stably transduced A549 cells protects against staurosporine- and Fas-induced depolarization of mitochondria and PARP cleavage.A. A549 cells were stably transduced with a lentivirus encoding RV capsid. Following induction with doxycycline (Dox), approximately <40% of cells were found to express capsid protein as detected by indirect immunofluorescence with a mouse monoclonal antibody and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. Cells expressing capsid (Cap) or luciferase (Luc) were treated with anti-Fas for 0, 2 and 6 after which levels of cleaved PARP (c-PARP) were determined by immunoblotting. Monitoring GAPDH levels was done to illustrate comparable loading. C. Capsid protein or luciferase expression was induced with doxycycline for 48 hours and then cells were treated with staurosporine (ST) or anti-Fas antibody for 6 hours to induce apoptosis. Samples were then stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. The relative level of specific cell death in each sample was calculated and plotted (D). Error bars indicate standard deviations. Student's t-Test was performed to determine statistical significance *p≤0.01 **p≤0.005.
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ppat-1001291-g003: Expression of capsid in stably transduced A549 cells protects against staurosporine- and Fas-induced depolarization of mitochondria and PARP cleavage.A. A549 cells were stably transduced with a lentivirus encoding RV capsid. Following induction with doxycycline (Dox), approximately <40% of cells were found to express capsid protein as detected by indirect immunofluorescence with a mouse monoclonal antibody and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. Cells expressing capsid (Cap) or luciferase (Luc) were treated with anti-Fas for 0, 2 and 6 after which levels of cleaved PARP (c-PARP) were determined by immunoblotting. Monitoring GAPDH levels was done to illustrate comparable loading. C. Capsid protein or luciferase expression was induced with doxycycline for 48 hours and then cells were treated with staurosporine (ST) or anti-Fas antibody for 6 hours to induce apoptosis. Samples were then stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. The relative level of specific cell death in each sample was calculated and plotted (D). Error bars indicate standard deviations. Student's t-Test was performed to determine statistical significance *p≤0.01 **p≤0.005.

Mentions: We next endeavored to identify what step in apoptotic signaling was blocked by capsid protein. For these experiments, lentiviral transduction was used to create A549 cells that stably express capsid protein under the control of a doxycycline-regulated promoter. Results from indirect immunofluorescence showed that less than 50% of the polyclonal population of transduced cells expressed RV capsid following doxycyline treatment (Figure 3A). Similar to results shown in Figure 2, induction of capsid expression protected the stably transduced A549 cells against staurosporine- and Fas-mediated activation of caspase 3 (Figure S3). To further confirm that apoptotic stimuli do not activate caspases in these cells, we measured the appearance of the downstream caspase 3 substrate, cleaved Poly(ADP-ribose) polymerase (PARP). Figure 3B shows that expression of capsid protein results in decreased anti-Fas-induced cleavage of PARP compared to luciferase-expressing cells. These data indicate that capsid protects A549 cells from staurosporine and anti-Fas treatment by blocking caspase activation.


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)

Expression of capsid in stably transduced A549 cells protects against staurosporine- and Fas-induced depolarization of mitochondria and PARP cleavage.A. A549 cells were stably transduced with a lentivirus encoding RV capsid. Following induction with doxycycline (Dox), approximately <40% of cells were found to express capsid protein as detected by indirect immunofluorescence with a mouse monoclonal antibody and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. Cells expressing capsid (Cap) or luciferase (Luc) were treated with anti-Fas for 0, 2 and 6 after which levels of cleaved PARP (c-PARP) were determined by immunoblotting. Monitoring GAPDH levels was done to illustrate comparable loading. C. Capsid protein or luciferase expression was induced with doxycycline for 48 hours and then cells were treated with staurosporine (ST) or anti-Fas antibody for 6 hours to induce apoptosis. Samples were then stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. The relative level of specific cell death in each sample was calculated and plotted (D). Error bars indicate standard deviations. Student's t-Test was performed to determine statistical significance *p≤0.01 **p≤0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g003: Expression of capsid in stably transduced A549 cells protects against staurosporine- and Fas-induced depolarization of mitochondria and PARP cleavage.A. A549 cells were stably transduced with a lentivirus encoding RV capsid. Following induction with doxycycline (Dox), approximately <40% of cells were found to express capsid protein as detected by indirect immunofluorescence with a mouse monoclonal antibody and chicken anti-mouse Alexa594. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. Cells expressing capsid (Cap) or luciferase (Luc) were treated with anti-Fas for 0, 2 and 6 after which levels of cleaved PARP (c-PARP) were determined by immunoblotting. Monitoring GAPDH levels was done to illustrate comparable loading. C. Capsid protein or luciferase expression was induced with doxycycline for 48 hours and then cells were treated with staurosporine (ST) or anti-Fas antibody for 6 hours to induce apoptosis. Samples were then stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. The relative level of specific cell death in each sample was calculated and plotted (D). Error bars indicate standard deviations. Student's t-Test was performed to determine statistical significance *p≤0.01 **p≤0.005.
Mentions: We next endeavored to identify what step in apoptotic signaling was blocked by capsid protein. For these experiments, lentiviral transduction was used to create A549 cells that stably express capsid protein under the control of a doxycycline-regulated promoter. Results from indirect immunofluorescence showed that less than 50% of the polyclonal population of transduced cells expressed RV capsid following doxycyline treatment (Figure 3A). Similar to results shown in Figure 2, induction of capsid expression protected the stably transduced A549 cells against staurosporine- and Fas-mediated activation of caspase 3 (Figure S3). To further confirm that apoptotic stimuli do not activate caspases in these cells, we measured the appearance of the downstream caspase 3 substrate, cleaved Poly(ADP-ribose) polymerase (PARP). Figure 3B shows that expression of capsid protein results in decreased anti-Fas-induced cleavage of PARP compared to luciferase-expressing cells. These data indicate that capsid protects A549 cells from staurosporine and anti-Fas treatment by blocking caspase activation.

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