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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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The arginine-rich motif in capsid protein is not required for targeting to mitochondria or activation of Bax.A. In the CapC5RA mutant, 5 arginine residues in the R motif were changed to alanines. RNA  =  RNA binding site; R =  arginine-rich motif; SP  =  E2 signal peptide. B. A549 cells were transfected with plasmids encoding wild type capsid or CapC5RA. At 40 hours post-transfection, cells were processed for indirect immunofluorescence using goat anti-RV, rabbit anti-cytochrome c and a mouse monoclonal antibody specific for activated Bax (6A7). Asterisk denotes a cell expressing CapC5RA that has loss its mitochondrial stores of cytochrome c. C. Samples were processed as in panel B except that a mouse monoclonal antibody to Complex II was included (instead of anti-Bax) to show that CapCR5A was targeted to mitochondria. Scale bars  = 10 µm.
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ppat-1001291-g010: The arginine-rich motif in capsid protein is not required for targeting to mitochondria or activation of Bax.A. In the CapC5RA mutant, 5 arginine residues in the R motif were changed to alanines. RNA  =  RNA binding site; R =  arginine-rich motif; SP  =  E2 signal peptide. B. A549 cells were transfected with plasmids encoding wild type capsid or CapC5RA. At 40 hours post-transfection, cells were processed for indirect immunofluorescence using goat anti-RV, rabbit anti-cytochrome c and a mouse monoclonal antibody specific for activated Bax (6A7). Asterisk denotes a cell expressing CapC5RA that has loss its mitochondrial stores of cytochrome c. C. Samples were processed as in panel B except that a mouse monoclonal antibody to Complex II was included (instead of anti-Bax) to show that CapCR5A was targeted to mitochondria. Scale bars  = 10 µm.

Mentions: To investigate if the arginine residues in the membrane-proximal R motif were important for the anti-apoptitic function of capsid protein, we created a point mutant (CapCR5A) in which five arginines in this motif were changed to alanine residues (Figure 10A). This capsid mutant was targeted to mitochondria where it activated Bax and stimulated cytochrome c release in the absence of apoptotic stimuli (Figure 10B and C asterisks); indicating that the arginine residues within the R domain are critical for the anti-apoptotic activity of capsid protein. Moreover, it would appear that mutation of these arginine residues unmasks an intrinsic pro-apoptotic activity of capsid protein, which may explain why it alone can stimulate Bax conformational change and membrane insertion. Next, we compared the Bax-binding ability of the CR5A mutant relative to wild type capsid and capsid deletion constructs. The observation that more CapΔSP is recovered in anti-Bax coimmunoprecipitations than CapΔRSP (Figure 11A) suggests that the R domain is important for interaction with Bax. However, ablation of the arginine residues in the R domain did not affect binding to Bax indicating that the arginine residues per se in this motif are not essential for interaction with Bax (Figure 11A). Binding between Bax and CapCT or CapNT was not detected in our assays (Figure 11B). Indirect immunofluorescence analyses revealed that unlike wild type capsid and CapCR5A, neither CapNT, CapCT, CapΔSP nor CapΔRSP induced the 6A7-specific conformation change in Bax (data not shown). Together, these results suggest that capsid protein employs a multi-step mechanism to block apoptosis. Specifically, binding to Bax through the R domain and/or the carboxyl terminus stimulates a conformational change in Bax; but pore formation and/or functionality is blocked by the arginines in the R motif of capsid protein.


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)

The arginine-rich motif in capsid protein is not required for targeting to mitochondria or activation of Bax.A. In the CapC5RA mutant, 5 arginine residues in the R motif were changed to alanines. RNA  =  RNA binding site; R =  arginine-rich motif; SP  =  E2 signal peptide. B. A549 cells were transfected with plasmids encoding wild type capsid or CapC5RA. At 40 hours post-transfection, cells were processed for indirect immunofluorescence using goat anti-RV, rabbit anti-cytochrome c and a mouse monoclonal antibody specific for activated Bax (6A7). Asterisk denotes a cell expressing CapC5RA that has loss its mitochondrial stores of cytochrome c. C. Samples were processed as in panel B except that a mouse monoclonal antibody to Complex II was included (instead of anti-Bax) to show that CapCR5A was targeted to mitochondria. Scale bars  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g010: The arginine-rich motif in capsid protein is not required for targeting to mitochondria or activation of Bax.A. In the CapC5RA mutant, 5 arginine residues in the R motif were changed to alanines. RNA  =  RNA binding site; R =  arginine-rich motif; SP  =  E2 signal peptide. B. A549 cells were transfected with plasmids encoding wild type capsid or CapC5RA. At 40 hours post-transfection, cells were processed for indirect immunofluorescence using goat anti-RV, rabbit anti-cytochrome c and a mouse monoclonal antibody specific for activated Bax (6A7). Asterisk denotes a cell expressing CapC5RA that has loss its mitochondrial stores of cytochrome c. C. Samples were processed as in panel B except that a mouse monoclonal antibody to Complex II was included (instead of anti-Bax) to show that CapCR5A was targeted to mitochondria. Scale bars  = 10 µm.
Mentions: To investigate if the arginine residues in the membrane-proximal R motif were important for the anti-apoptitic function of capsid protein, we created a point mutant (CapCR5A) in which five arginines in this motif were changed to alanine residues (Figure 10A). This capsid mutant was targeted to mitochondria where it activated Bax and stimulated cytochrome c release in the absence of apoptotic stimuli (Figure 10B and C asterisks); indicating that the arginine residues within the R domain are critical for the anti-apoptotic activity of capsid protein. Moreover, it would appear that mutation of these arginine residues unmasks an intrinsic pro-apoptotic activity of capsid protein, which may explain why it alone can stimulate Bax conformational change and membrane insertion. Next, we compared the Bax-binding ability of the CR5A mutant relative to wild type capsid and capsid deletion constructs. The observation that more CapΔSP is recovered in anti-Bax coimmunoprecipitations than CapΔRSP (Figure 11A) suggests that the R domain is important for interaction with Bax. However, ablation of the arginine residues in the R domain did not affect binding to Bax indicating that the arginine residues per se in this motif are not essential for interaction with Bax (Figure 11A). Binding between Bax and CapCT or CapNT was not detected in our assays (Figure 11B). Indirect immunofluorescence analyses revealed that unlike wild type capsid and CapCR5A, neither CapNT, CapCT, CapΔSP nor CapΔRSP induced the 6A7-specific conformation change in Bax (data not shown). Together, these results suggest that capsid protein employs a multi-step mechanism to block apoptosis. Specifically, binding to Bax through the R domain and/or the carboxyl terminus stimulates a conformational change in Bax; but pore formation and/or functionality is blocked by the arginines in the R motif of capsid protein.

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