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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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Capsid protein forms complexes with Bax.A. Stably transduced A549 cells expressing luciferase (Luc) or capsid protein (Cap) were treated with staurosporine (ST) for 6 hours. After ST treatment, crude mitochondria were isolated and then crosslinked with BMH prior to analyses by immunblotting with antibodies to capsid and Bax. The arrowhead in the upper panels denotes an SDS-resistant Bax dimer that is present in all samples. The asterisk in the lower panels denotes a non-specific protein that cross-reacts with the capsid antibody. A549 cells were infected with RV and at 42 hours, were treated with ST for 0 to 6 hours. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to capsid, Bax (B) or Bak (C) followed by SDS-PAGE and immunoblotting (IB).
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ppat-1001291-g005: Capsid protein forms complexes with Bax.A. Stably transduced A549 cells expressing luciferase (Luc) or capsid protein (Cap) were treated with staurosporine (ST) for 6 hours. After ST treatment, crude mitochondria were isolated and then crosslinked with BMH prior to analyses by immunblotting with antibodies to capsid and Bax. The arrowhead in the upper panels denotes an SDS-resistant Bax dimer that is present in all samples. The asterisk in the lower panels denotes a non-specific protein that cross-reacts with the capsid antibody. A549 cells were infected with RV and at 42 hours, were treated with ST for 0 to 6 hours. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to capsid, Bax (B) or Bak (C) followed by SDS-PAGE and immunoblotting (IB).

Mentions: Bax and Bak are two key apoptotic molecules that form oligomers on mitochondria [24], [25], [26] and apoptosis occurs when the mitochondrial outer membrane is permeabilized by these pore-forming molecules [27]. Accordingly, we next focused our efforts on these Bcl-2 family members starting with Bax. Normally, Bax is an inactive monomer found in the cytosol or loosely bound to the mitochondrial outer membrane of healthy cells [28], [29]. In response to apoptotic stimuli, Bax activation is characterized by a multi-step process whereby it undergoes a conformational change [30], [31], integrates into the mitochondrial membrane [28], [32] where it forms higher order oligomers [33]. It is the large Bax oligomers that are linked to the formation of membrane pores that facilitate release of mitochondrial cytochrome c and downstream caspase activation [33], [34]. Of these multiple steps, Bax conformational change can be detected by immunoreactivity with a conformation-specific antibody, 6A7 [35], [36]. We observed that RV infection induces Bax conformational change, however cytochrome c remained associated with mitochondria (Figure 4A, arrows). Moreover, Bax conformational change as detected by 6A7 staining was evident in the majority (76%) of cells expressing capsid protein (Figure 4B arrows). In contrast, among cells expressing the viral glycoproteins E2 and E1, only 6% contained activated Bax. Despite initial stimulation of Bax, similar to infected cells, no loss of cytochrome c from mitochondria was observed in capsid-expressing cells. Because capsid protein stimulates Bax in a manner that does not produce functional pores that mediate efflux of cytochrome c, we initially thought that capsid protein blocks oligomerization of Bax. However, data in Figure 5A indicate that this is not the case. Rather, our results suggest that capsid protein and Bax form mixed large hetero-oligomers even in the absence of apoptotic stimuli. Indeed, reciprocal co-immunoprecipitation experiments confirmed that capsid forms a stable complex with Bax (Figure 5B). Staurosporine treatment enhanced the formation of the capsid:Bax hetero-oligomers but evidently did not facilitate the assembly of functional Bax pores as the cells were not apoptotic. Interestingly, we found no evidence that capsid protein binds to Bak (Figure 5C) suggesting the interaction of this viral protein with Bcl-2 family proteins is highly specific. Together, these data suggest that capsid protein and Bax form mixed oligomers that do not function as pores.


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)

Capsid protein forms complexes with Bax.A. Stably transduced A549 cells expressing luciferase (Luc) or capsid protein (Cap) were treated with staurosporine (ST) for 6 hours. After ST treatment, crude mitochondria were isolated and then crosslinked with BMH prior to analyses by immunblotting with antibodies to capsid and Bax. The arrowhead in the upper panels denotes an SDS-resistant Bax dimer that is present in all samples. The asterisk in the lower panels denotes a non-specific protein that cross-reacts with the capsid antibody. A549 cells were infected with RV and at 42 hours, were treated with ST for 0 to 6 hours. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to capsid, Bax (B) or Bak (C) followed by SDS-PAGE and immunoblotting (IB).
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Related In: Results  -  Collection

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ppat-1001291-g005: Capsid protein forms complexes with Bax.A. Stably transduced A549 cells expressing luciferase (Luc) or capsid protein (Cap) were treated with staurosporine (ST) for 6 hours. After ST treatment, crude mitochondria were isolated and then crosslinked with BMH prior to analyses by immunblotting with antibodies to capsid and Bax. The arrowhead in the upper panels denotes an SDS-resistant Bax dimer that is present in all samples. The asterisk in the lower panels denotes a non-specific protein that cross-reacts with the capsid antibody. A549 cells were infected with RV and at 42 hours, were treated with ST for 0 to 6 hours. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to capsid, Bax (B) or Bak (C) followed by SDS-PAGE and immunoblotting (IB).
Mentions: Bax and Bak are two key apoptotic molecules that form oligomers on mitochondria [24], [25], [26] and apoptosis occurs when the mitochondrial outer membrane is permeabilized by these pore-forming molecules [27]. Accordingly, we next focused our efforts on these Bcl-2 family members starting with Bax. Normally, Bax is an inactive monomer found in the cytosol or loosely bound to the mitochondrial outer membrane of healthy cells [28], [29]. In response to apoptotic stimuli, Bax activation is characterized by a multi-step process whereby it undergoes a conformational change [30], [31], integrates into the mitochondrial membrane [28], [32] where it forms higher order oligomers [33]. It is the large Bax oligomers that are linked to the formation of membrane pores that facilitate release of mitochondrial cytochrome c and downstream caspase activation [33], [34]. Of these multiple steps, Bax conformational change can be detected by immunoreactivity with a conformation-specific antibody, 6A7 [35], [36]. We observed that RV infection induces Bax conformational change, however cytochrome c remained associated with mitochondria (Figure 4A, arrows). Moreover, Bax conformational change as detected by 6A7 staining was evident in the majority (76%) of cells expressing capsid protein (Figure 4B arrows). In contrast, among cells expressing the viral glycoproteins E2 and E1, only 6% contained activated Bax. Despite initial stimulation of Bax, similar to infected cells, no loss of cytochrome c from mitochondria was observed in capsid-expressing cells. Because capsid protein stimulates Bax in a manner that does not produce functional pores that mediate efflux of cytochrome c, we initially thought that capsid protein blocks oligomerization of Bax. However, data in Figure 5A indicate that this is not the case. Rather, our results suggest that capsid protein and Bax form mixed large hetero-oligomers even in the absence of apoptotic stimuli. Indeed, reciprocal co-immunoprecipitation experiments confirmed that capsid forms a stable complex with Bax (Figure 5B). Staurosporine treatment enhanced the formation of the capsid:Bax hetero-oligomers but evidently did not facilitate the assembly of functional Bax pores as the cells were not apoptotic. Interestingly, we found no evidence that capsid protein binds to Bak (Figure 5C) suggesting the interaction of this viral protein with Bcl-2 family proteins is highly specific. Together, these data suggest that capsid protein and Bax form mixed oligomers that do not function as pores.

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