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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 carboxyl-terminal R motif in capsid protein is required to block apoptosis during RV infection.A. A549 cells were infected with wild type (WT) or CR5A strains of RV (MOI = 1) and 42 hours later, cells were treated with anti-Fas to induce apoptosis. Cells were processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. The average number of double-positive cells for each experimental condition was determined from three independent experiments and the results were plotted. *p≤0.005, **p≤0.001. B. Vero cells were infected with WT or CR5A strains of RV (MOI = 1). At the indicated times, cell lysates (60 µg) were subjected to immunoblot analyses using antibodies to p150, capsid, E1 and GAPDH (loading control). C. Cell culture supernatants from infected Vero cells were centrifuged at 100,000× g and the pellets were subjected to immunoblot analyses using antibodies to capsid. D. Relative levels of genomic viral RNA (gRNA) in the infected Vero cells were determined by qRT-PCR at the indicated time periods. E. RK13 cells were infected with wild type WT or CR5A strains of RV and plaque assays were performed. The CR5A-derived plaques are much larger and have a spotty appearance.
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ppat-1001291-g012: The carboxyl-terminal R motif in capsid protein is required to block apoptosis during RV infection.A. A549 cells were infected with wild type (WT) or CR5A strains of RV (MOI = 1) and 42 hours later, cells were treated with anti-Fas to induce apoptosis. Cells were processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. The average number of double-positive cells for each experimental condition was determined from three independent experiments and the results were plotted. *p≤0.005, **p≤0.001. B. Vero cells were infected with WT or CR5A strains of RV (MOI = 1). At the indicated times, cell lysates (60 µg) were subjected to immunoblot analyses using antibodies to p150, capsid, E1 and GAPDH (loading control). C. Cell culture supernatants from infected Vero cells were centrifuged at 100,000× g and the pellets were subjected to immunoblot analyses using antibodies to capsid. D. Relative levels of genomic viral RNA (gRNA) in the infected Vero cells were determined by qRT-PCR at the indicated time periods. E. RK13 cells were infected with wild type WT or CR5A strains of RV and plaque assays were performed. The CR5A-derived plaques are much larger and have a spotty appearance.

Mentions: We introduced the CR5A mutations into the capsid gene of a RV infectious clone in order to determine if the membrane-proximal arginine-rich (R) domain in capsid protein is required for blocking apoptosis during infection. Our hypothesis was that early onset apoptosis would result in decreased replication and virus particle production. A549 cells were infected with wild type or CR5A strains of RV and virus replication and apoptosis induction were analyzed. Data in Figure 12A show that cells infected with CR5A virus were significantly more susceptible to Fas-dependent apoptosis. Moreover, in non-treated (control) samples, the level of virus-induced apoptosis was four fold higher in cells infected with the CR5A mutant. Similar results were obtained with infected Vero cells (data not shown). Next, we compared the levels of RV proteins in CR5A and wild type (WT) RV infected cells as a function of time. Figure 12B shows that in cells that were infected with WT RV, the level of virus nonstructural (p150) and structural proteins (capsid) peaked at 72 hours. In contrast to p150 levels which were only moderately lower, steady state levels of capsid protein were dramatically lower in CR5A infected cells at all time points. To control for the possibility that CR5A capsid was unstable in the infected cells, we also determined the relative levels of another structural protein, E1. Similar to capsid protein levels in CR5A infected cells, levels of E1 were much lower than in WT virus infected cells; suggesting a defect in synthesis of structural proteins in CR5A infected cells. Consistent with this theory, data in Figure 12C show that secretion of CR5A virions is severly impaired. This was not because the CR5A capsid is misfolded as data in Figure S5 show that this mutant capsid protein functions as well as wild type capsid in driving assembly and secretion of Rubella virus-like particles.


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 carboxyl-terminal R motif in capsid protein is required to block apoptosis during RV infection.A. A549 cells were infected with wild type (WT) or CR5A strains of RV (MOI = 1) and 42 hours later, cells were treated with anti-Fas to induce apoptosis. Cells were processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. The average number of double-positive cells for each experimental condition was determined from three independent experiments and the results were plotted. *p≤0.005, **p≤0.001. B. Vero cells were infected with WT or CR5A strains of RV (MOI = 1). At the indicated times, cell lysates (60 µg) were subjected to immunoblot analyses using antibodies to p150, capsid, E1 and GAPDH (loading control). C. Cell culture supernatants from infected Vero cells were centrifuged at 100,000× g and the pellets were subjected to immunoblot analyses using antibodies to capsid. D. Relative levels of genomic viral RNA (gRNA) in the infected Vero cells were determined by qRT-PCR at the indicated time periods. E. RK13 cells were infected with wild type WT or CR5A strains of RV and plaque assays were performed. The CR5A-derived plaques are much larger and have a spotty appearance.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g012: The carboxyl-terminal R motif in capsid protein is required to block apoptosis during RV infection.A. A549 cells were infected with wild type (WT) or CR5A strains of RV (MOI = 1) and 42 hours later, cells were treated with anti-Fas to induce apoptosis. Cells were processed for indirect immunofluorescence using rabbit anti-caspase 3 and mouse anti-capsid. The average number of double-positive cells for each experimental condition was determined from three independent experiments and the results were plotted. *p≤0.005, **p≤0.001. B. Vero cells were infected with WT or CR5A strains of RV (MOI = 1). At the indicated times, cell lysates (60 µg) were subjected to immunoblot analyses using antibodies to p150, capsid, E1 and GAPDH (loading control). C. Cell culture supernatants from infected Vero cells were centrifuged at 100,000× g and the pellets were subjected to immunoblot analyses using antibodies to capsid. D. Relative levels of genomic viral RNA (gRNA) in the infected Vero cells were determined by qRT-PCR at the indicated time periods. E. RK13 cells were infected with wild type WT or CR5A strains of RV and plaque assays were performed. The CR5A-derived plaques are much larger and have a spotty appearance.
Mentions: We introduced the CR5A mutations into the capsid gene of a RV infectious clone in order to determine if the membrane-proximal arginine-rich (R) domain in capsid protein is required for blocking apoptosis during infection. Our hypothesis was that early onset apoptosis would result in decreased replication and virus particle production. A549 cells were infected with wild type or CR5A strains of RV and virus replication and apoptosis induction were analyzed. Data in Figure 12A show that cells infected with CR5A virus were significantly more susceptible to Fas-dependent apoptosis. Moreover, in non-treated (control) samples, the level of virus-induced apoptosis was four fold higher in cells infected with the CR5A mutant. Similar results were obtained with infected Vero cells (data not shown). Next, we compared the levels of RV proteins in CR5A and wild type (WT) RV infected cells as a function of time. Figure 12B shows that in cells that were infected with WT RV, the level of virus nonstructural (p150) and structural proteins (capsid) peaked at 72 hours. In contrast to p150 levels which were only moderately lower, steady state levels of capsid protein were dramatically lower in CR5A infected cells at all time points. To control for the possibility that CR5A capsid was unstable in the infected cells, we also determined the relative levels of another structural protein, E1. Similar to capsid protein levels in CR5A infected cells, levels of E1 were much lower than in WT virus infected cells; suggesting a defect in synthesis of structural proteins in CR5A infected cells. Consistent with this theory, data in Figure 12C show that secretion of CR5A virions is severly impaired. This was not because the CR5A capsid is misfolded as data in Figure S5 show that this mutant capsid protein functions as well as wild type capsid in driving assembly and secretion of Rubella virus-like particles.

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