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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

Vero cells that were infected with the CR5A mutant or wild type RV were treated with DMSO (-) or Z-VAD-FMK (50 µM) every day for up to 3 days.A. Cell lysates and culture supernatants were harvested at indicated time periods. Equal amounts of cell lysates (60 µg) were subjected to immunoblot analyses with antibodies to viral (p150 and capsid) and cellular proteins (GAPDH). At each time point, the relative signals of the p150/GAPDH and capsid/GAPDH in DMSO-treated cells were normalized to 1.0. Relative ratios of Z-VAD-FMK to DMSO samples were then determined. In some cases (ND), it was not possible to determine the relative ratios due to low signal intensity. B. Media from infected cells were serially diluted 10-fold before addition to RK-13 monolayers for plaque assays. Based on the clearing of cell monolayers, it can be seen that supernatants from CR5A infected Z-VAD-FMK-treated Vero cells contain more infectious virus.
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ppat-1001291-g013: Vero cells that were infected with the CR5A mutant or wild type RV were treated with DMSO (-) or Z-VAD-FMK (50 µM) every day for up to 3 days.A. Cell lysates and culture supernatants were harvested at indicated time periods. Equal amounts of cell lysates (60 µg) were subjected to immunoblot analyses with antibodies to viral (p150 and capsid) and cellular proteins (GAPDH). At each time point, the relative signals of the p150/GAPDH and capsid/GAPDH in DMSO-treated cells were normalized to 1.0. Relative ratios of Z-VAD-FMK to DMSO samples were then determined. In some cases (ND), it was not possible to determine the relative ratios due to low signal intensity. B. Media from infected cells were serially diluted 10-fold before addition to RK-13 monolayers for plaque assays. Based on the clearing of cell monolayers, it can be seen that supernatants from CR5A infected Z-VAD-FMK-treated Vero cells contain more infectious virus.

Mentions: Although data in Figure S5 indicate that Cap5RA is not misfolded, without additional investigation, we could not completely rule out the possibility that the replication defects associated with the CR5A strain virus were due to other inherent defects of the mutant capsid protein. Therefore, we attempted to artificially block apoptosis by over-expression of Bcl-XL or adding the caspase inhibitor Z-VAD-FMK to CR5A infected cells. Over-expression of Bcl-XL did not rescue the CR5A replication but this result was non-informative as further investigation revealed that this anti-apoptotic protein was unable to protect mitochondria from the effects of CapC5RA in transfected cells (data not shown). In contrast, addition of Z-VAD-FMK did have a modest effect on production of viral proteins in CR5A infected cells (Figure 13A). The effect was most pronounced at 72 hrs post-infection where levels of p150 and capsid were considerably higher in Z-VAD-FMK treated cells. In contrast, blocking caspase activity in cells that were infected with wild type RV did not appreciably alter the steady state levels of viral proteins. Finally, it can be seen from the data in Figure 13B that Z-VAD-FMK treatment had a modest effect on production of CR5A virus. Compared to CR5A-infected Vero cells treated with DMSO alone, addition of Z-VAD-FMK resulted in a modest increase in viral titers as evidenced by increased clearing of RK-13 monolayers. Together, these data are consistent with our hypothesis that the anti-apoptotic function of capsid is important for virus replication.


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)

Vero cells that were infected with the CR5A mutant or wild type RV were treated with DMSO (-) or Z-VAD-FMK (50 µM) every day for up to 3 days.A. Cell lysates and culture supernatants were harvested at indicated time periods. Equal amounts of cell lysates (60 µg) were subjected to immunoblot analyses with antibodies to viral (p150 and capsid) and cellular proteins (GAPDH). At each time point, the relative signals of the p150/GAPDH and capsid/GAPDH in DMSO-treated cells were normalized to 1.0. Relative ratios of Z-VAD-FMK to DMSO samples were then determined. In some cases (ND), it was not possible to determine the relative ratios due to low signal intensity. B. Media from infected cells were serially diluted 10-fold before addition to RK-13 monolayers for plaque assays. Based on the clearing of cell monolayers, it can be seen that supernatants from CR5A infected Z-VAD-FMK-treated Vero cells contain more infectious virus.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001291-g013: Vero cells that were infected with the CR5A mutant or wild type RV were treated with DMSO (-) or Z-VAD-FMK (50 µM) every day for up to 3 days.A. Cell lysates and culture supernatants were harvested at indicated time periods. Equal amounts of cell lysates (60 µg) were subjected to immunoblot analyses with antibodies to viral (p150 and capsid) and cellular proteins (GAPDH). At each time point, the relative signals of the p150/GAPDH and capsid/GAPDH in DMSO-treated cells were normalized to 1.0. Relative ratios of Z-VAD-FMK to DMSO samples were then determined. In some cases (ND), it was not possible to determine the relative ratios due to low signal intensity. B. Media from infected cells were serially diluted 10-fold before addition to RK-13 monolayers for plaque assays. Based on the clearing of cell monolayers, it can be seen that supernatants from CR5A infected Z-VAD-FMK-treated Vero cells contain more infectious virus.
Mentions: Although data in Figure S5 indicate that Cap5RA is not misfolded, without additional investigation, we could not completely rule out the possibility that the replication defects associated with the CR5A strain virus were due to other inherent defects of the mutant capsid protein. Therefore, we attempted to artificially block apoptosis by over-expression of Bcl-XL or adding the caspase inhibitor Z-VAD-FMK to CR5A infected cells. Over-expression of Bcl-XL did not rescue the CR5A replication but this result was non-informative as further investigation revealed that this anti-apoptotic protein was unable to protect mitochondria from the effects of CapC5RA in transfected cells (data not shown). In contrast, addition of Z-VAD-FMK did have a modest effect on production of viral proteins in CR5A infected cells (Figure 13A). The effect was most pronounced at 72 hrs post-infection where levels of p150 and capsid were considerably higher in Z-VAD-FMK treated cells. In contrast, blocking caspase activity in cells that were infected with wild type RV did not appreciably alter the steady state levels of viral proteins. Finally, it can be seen from the data in Figure 13B that Z-VAD-FMK treatment had a modest effect on production of CR5A virus. Compared to CR5A-infected Vero cells treated with DMSO alone, addition of Z-VAD-FMK resulted in a modest increase in viral titers as evidenced by increased clearing of RK-13 monolayers. Together, these data are consistent with our hypothesis that the anti-apoptotic function of capsid is important for virus replication.

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