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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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Related in: MedlinePlus

Capsid expression protects against Bax- but not Bak-induced apoptosis.A549 cells were co-transfected with plasmids encoding GFP-Bax or GFP-Bak together with plasmids encoding capsid, CapNT, Bcl-XL (not shown) or vector alone. After 24 hours, samples were stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. Sample FACS plots for GFP-Bax (A) and GFP-Bak (B) transfectants are shown. C. The levels of relative specific cell death in GFP positive cells were calculated and plotted. Error bars indicate standard deviations calculated from three independent experiments. *p≤0.001, **p≤0.01.
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ppat-1001291-g006: Capsid expression protects against Bax- but not Bak-induced apoptosis.A549 cells were co-transfected with plasmids encoding GFP-Bax or GFP-Bak together with plasmids encoding capsid, CapNT, Bcl-XL (not shown) or vector alone. After 24 hours, samples were stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. Sample FACS plots for GFP-Bax (A) and GFP-Bak (B) transfectants are shown. C. The levels of relative specific cell death in GFP positive cells were calculated and plotted. Error bars indicate standard deviations calculated from three independent experiments. *p≤0.001, **p≤0.01.

Mentions: Since capsid protein forms a complex with Bax, we next tested whether its expression could inhibit Bax-mediated apoptosis. Over-expression of either Bax or Bak induces cell death in the absence of other apoptotic stimuli [37]. A549 cells were co-transfected with plasmids encoding GFP-Bax and capsid, Bcl-XL (positive control) or vector alone (negative control) and at 24 hour post-transfection, samples were stained with the membrane-potential specific dye TMRM and then subjected to flow cytometric analyses (Figure 6A). As a second control, we transfected cells with a plasmid encoding a capsid deletion construct (CapNT) that is not targeted to mitochondria (see below). Loss of TMRM staining as a result of depolarization of mitochondrial membranes was used as the measure of apoptotic cell death. Quantitation of the data (Figure 6C) revealed that expression of capsid protein reduced the level of Bax-induced cell death by more than 60% compared to CapNT or vector alone. Similar results were observed for cells expressing Bcl-XL, a protein which has previously been shown to block the effects of Bax over-expression [38]. Data in Figure S4 show that capsid expression also protects primary human embryonic fibroblast (HEL-18) cells [17] from Bax-mediated apoptosis. The anti-apoptotic activity of capsid protein was specific to Bax as evidenced the fact that it did not attenuate Bak-mediated apoptosis (Figure 6B, C).


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 expression protects against Bax- but not Bak-induced apoptosis.A549 cells were co-transfected with plasmids encoding GFP-Bax or GFP-Bak together with plasmids encoding capsid, CapNT, Bcl-XL (not shown) or vector alone. After 24 hours, samples were stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. Sample FACS plots for GFP-Bax (A) and GFP-Bak (B) transfectants are shown. C. The levels of relative specific cell death in GFP positive cells were calculated and plotted. Error bars indicate standard deviations calculated from three independent experiments. *p≤0.001, **p≤0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g006: Capsid expression protects against Bax- but not Bak-induced apoptosis.A549 cells were co-transfected with plasmids encoding GFP-Bax or GFP-Bak together with plasmids encoding capsid, CapNT, Bcl-XL (not shown) or vector alone. After 24 hours, samples were stained with TMRM for 30 minutes and then subjected to flow cytometric analyses. Sample FACS plots for GFP-Bax (A) and GFP-Bak (B) transfectants are shown. C. The levels of relative specific cell death in GFP positive cells were calculated and plotted. Error bars indicate standard deviations calculated from three independent experiments. *p≤0.001, **p≤0.01.
Mentions: Since capsid protein forms a complex with Bax, we next tested whether its expression could inhibit Bax-mediated apoptosis. Over-expression of either Bax or Bak induces cell death in the absence of other apoptotic stimuli [37]. A549 cells were co-transfected with plasmids encoding GFP-Bax and capsid, Bcl-XL (positive control) or vector alone (negative control) and at 24 hour post-transfection, samples were stained with the membrane-potential specific dye TMRM and then subjected to flow cytometric analyses (Figure 6A). As a second control, we transfected cells with a plasmid encoding a capsid deletion construct (CapNT) that is not targeted to mitochondria (see below). Loss of TMRM staining as a result of depolarization of mitochondrial membranes was used as the measure of apoptotic cell death. Quantitation of the data (Figure 6C) revealed that expression of capsid protein reduced the level of Bax-induced cell death by more than 60% compared to CapNT or vector alone. Similar results were observed for cells expressing Bcl-XL, a protein which has previously been shown to block the effects of Bax over-expression [38]. Data in Figure S4 show that capsid expression also protects primary human embryonic fibroblast (HEL-18) cells [17] from Bax-mediated apoptosis. The anti-apoptotic activity of capsid protein was specific to Bax as evidenced the fact that it did not attenuate Bak-mediated apoptosis (Figure 6B, C).

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