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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 RV capsid protein blocks activation of caspase 3.A. A549 cells were transiently transfected with plasmids encoding eGFP, RV glycoproteins E2 and E1, capsid protein or the anti-apoptotic protein Bcl-XL. At forty hours post-transfection, cells were treated with anti-Fas for 6 hours after which time they were processed for immunofluorescence using rabbit anti-caspase 3 and mouse antibodies to E1, capsid or Bcl-XL. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. For samples expressing eGFP, the rabbit anti-caspase 3 was detected with donkey anti-rabbit conjugated to Texas Red. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. The percentages of transfected cells expressing active caspase 3 (double positive) were determined and plotted. Error bars indicate standard deviations calculated from three independent experiments in which at least 100 cells for each experiment were scored. One-way ANOVA was used to determine statistical significance. p = ≤0.001.
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ppat-1001291-g002: Expression of RV capsid protein blocks activation of caspase 3.A. A549 cells were transiently transfected with plasmids encoding eGFP, RV glycoproteins E2 and E1, capsid protein or the anti-apoptotic protein Bcl-XL. At forty hours post-transfection, cells were treated with anti-Fas for 6 hours after which time they were processed for immunofluorescence using rabbit anti-caspase 3 and mouse antibodies to E1, capsid or Bcl-XL. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. For samples expressing eGFP, the rabbit anti-caspase 3 was detected with donkey anti-rabbit conjugated to Texas Red. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. The percentages of transfected cells expressing active caspase 3 (double positive) were determined and plotted. Error bars indicate standard deviations calculated from three independent experiments in which at least 100 cells for each experiment were scored. One-way ANOVA was used to determine statistical significance. p = ≤0.001.

Mentions: Next, we sought to determine which viral protein(s) was primarily responsible for protecting infected cells against apoptosis. Previous studies have indicated that expression of the nonstructural proteins p150 and p90 are cytotoxic [19], [20] and therefore, we focused our attention on the virus structural proteins. Plasmids encoding glycoproteins E2 and E1 or capsid, were transiently tranfected into A549 cells and at 40 hours post-transfection, cells were induced to undergo apoptosis by treatment with anti-Fas. Samples were processed for indirect immunofluorescence (Figure 2A) and the numbers of active caspase 3-positive transfectants were determined. Data in Figure 2B show that the levels of apoptosis were similar in cells expressing the viral glycoproteins E2 and E1 and the negative control protein eGFP. In contrast, expression of the RV capsid protein was just as protective against anti-Fas as the well-characterized anti-apoptotic protein Bcl-XL [21]. Compared to eGFP or E2E1 transfectants that were treated with anti-Fas, the percentage of apoptotic cells among capsid transfectants was three fold lower (Figure 2B).


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 RV capsid protein blocks activation of caspase 3.A. A549 cells were transiently transfected with plasmids encoding eGFP, RV glycoproteins E2 and E1, capsid protein or the anti-apoptotic protein Bcl-XL. At forty hours post-transfection, cells were treated with anti-Fas for 6 hours after which time they were processed for immunofluorescence using rabbit anti-caspase 3 and mouse antibodies to E1, capsid or Bcl-XL. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. For samples expressing eGFP, the rabbit anti-caspase 3 was detected with donkey anti-rabbit conjugated to Texas Red. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. The percentages of transfected cells expressing active caspase 3 (double positive) were determined and plotted. Error bars indicate standard deviations calculated from three independent experiments in which at least 100 cells for each experiment were scored. One-way ANOVA was used to determine statistical significance. p = ≤0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g002: Expression of RV capsid protein blocks activation of caspase 3.A. A549 cells were transiently transfected with plasmids encoding eGFP, RV glycoproteins E2 and E1, capsid protein or the anti-apoptotic protein Bcl-XL. At forty hours post-transfection, cells were treated with anti-Fas for 6 hours after which time they were processed for immunofluorescence using rabbit anti-caspase 3 and mouse antibodies to E1, capsid or Bcl-XL. Primary antibodies were detected with donkey anti-rabbit Alexa488 and chicken anti-mouse Alexa594. For samples expressing eGFP, the rabbit anti-caspase 3 was detected with donkey anti-rabbit conjugated to Texas Red. Nuclei were counter stained with DAPI. Scale bar  = 10 µm. B. The percentages of transfected cells expressing active caspase 3 (double positive) were determined and plotted. Error bars indicate standard deviations calculated from three independent experiments in which at least 100 cells for each experiment were scored. One-way ANOVA was used to determine statistical significance. p = ≤0.001.
Mentions: Next, we sought to determine which viral protein(s) was primarily responsible for protecting infected cells against apoptosis. Previous studies have indicated that expression of the nonstructural proteins p150 and p90 are cytotoxic [19], [20] and therefore, we focused our attention on the virus structural proteins. Plasmids encoding glycoproteins E2 and E1 or capsid, were transiently tranfected into A549 cells and at 40 hours post-transfection, cells were induced to undergo apoptosis by treatment with anti-Fas. Samples were processed for indirect immunofluorescence (Figure 2A) and the numbers of active caspase 3-positive transfectants were determined. Data in Figure 2B show that the levels of apoptosis were similar in cells expressing the viral glycoproteins E2 and E1 and the negative control protein eGFP. In contrast, expression of the RV capsid protein was just as protective against anti-Fas as the well-characterized anti-apoptotic protein Bcl-XL [21]. Compared to eGFP or E2E1 transfectants that were treated with anti-Fas, the percentage of apoptotic cells among capsid transfectants was three fold lower (Figure 2B).

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