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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 E2 signal peptide is required for targeting of capsid protein to mitochondria.A. Schematic of capsid constructs used for transfection experiments. RNA = RNA-binding domain; R = Arginine-rich motif; SP = E2 signal peptide. Numbers denote the amino acid residues of capsid protein. B. A549 cells were transfected with plasmids encoding capsid constructs and were then processed for indirect immunofluorescence after 40 hours. With the exception of transfectants expressing CapNT, all samples were stained with mouse anti-capsid and rabbit anti-cytochrome c to label mitochondria. Primary antibodies were detected with chicken anti-mouse Alexa594 and donkey anti-rabbit Alexa488. CapNT was detected using goat anti-RV serum and chicken anti-goat Alexa594. Nuclei were stained with DAPI. Scale bar  = 10 µm.
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ppat-1001291-g008: The E2 signal peptide is required for targeting of capsid protein to mitochondria.A. Schematic of capsid constructs used for transfection experiments. RNA = RNA-binding domain; R = Arginine-rich motif; SP = E2 signal peptide. Numbers denote the amino acid residues of capsid protein. B. A549 cells were transfected with plasmids encoding capsid constructs and were then processed for indirect immunofluorescence after 40 hours. With the exception of transfectants expressing CapNT, all samples were stained with mouse anti-capsid and rabbit anti-cytochrome c to label mitochondria. Primary antibodies were detected with chicken anti-mouse Alexa594 and donkey anti-rabbit Alexa488. CapNT was detected using goat anti-RV serum and chicken anti-goat Alexa594. Nuclei were stained with DAPI. Scale bar  = 10 µm.

Mentions: Based on the assumption that association of capsid protein with mitochondria is critical for its anti-apoptotic function, we next mapped the region of capsid protein that is required for targeting to this organelle. Analyses of the RV capsid protein sequence with web-based algorithms such as PSORT II Prediction (http://psort.nibb.ac.jp/form2.html) indicated that conventional mitochondrial targeting signals are absent. We therefore constructed a series of capsid deletion mutants whose localizations were determined by expression in A549 cells (Figure 8A). From the indirect immunofluorescence data shown in Figure 8B, it can be seen that the 23 amino acid residue E2 signal peptide which forms the hydrophobic carboxyl-terminus of capsid protein, is required for association with mitochondria. Moreover, the observation that a pool of CapCT overlaps with cytochrome c indicates that the carboxyl-terminal region of capsid protein contains information that is sufficient for targeting to mitochondria. Intriguingly, expression of the CapCT construct caused extreme compaction of the mitochondrial network to the perinuclear region, much more so than in cells expressing full-length 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 E2 signal peptide is required for targeting of capsid protein to mitochondria.A. Schematic of capsid constructs used for transfection experiments. RNA = RNA-binding domain; R = Arginine-rich motif; SP = E2 signal peptide. Numbers denote the amino acid residues of capsid protein. B. A549 cells were transfected with plasmids encoding capsid constructs and were then processed for indirect immunofluorescence after 40 hours. With the exception of transfectants expressing CapNT, all samples were stained with mouse anti-capsid and rabbit anti-cytochrome c to label mitochondria. Primary antibodies were detected with chicken anti-mouse Alexa594 and donkey anti-rabbit Alexa488. CapNT was detected using goat anti-RV serum and chicken anti-goat Alexa594. Nuclei were stained with DAPI. Scale bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040668&req=5

ppat-1001291-g008: The E2 signal peptide is required for targeting of capsid protein to mitochondria.A. Schematic of capsid constructs used for transfection experiments. RNA = RNA-binding domain; R = Arginine-rich motif; SP = E2 signal peptide. Numbers denote the amino acid residues of capsid protein. B. A549 cells were transfected with plasmids encoding capsid constructs and were then processed for indirect immunofluorescence after 40 hours. With the exception of transfectants expressing CapNT, all samples were stained with mouse anti-capsid and rabbit anti-cytochrome c to label mitochondria. Primary antibodies were detected with chicken anti-mouse Alexa594 and donkey anti-rabbit Alexa488. CapNT was detected using goat anti-RV serum and chicken anti-goat Alexa594. Nuclei were stained with DAPI. Scale bar  = 10 µm.
Mentions: Based on the assumption that association of capsid protein with mitochondria is critical for its anti-apoptotic function, we next mapped the region of capsid protein that is required for targeting to this organelle. Analyses of the RV capsid protein sequence with web-based algorithms such as PSORT II Prediction (http://psort.nibb.ac.jp/form2.html) indicated that conventional mitochondrial targeting signals are absent. We therefore constructed a series of capsid deletion mutants whose localizations were determined by expression in A549 cells (Figure 8A). From the indirect immunofluorescence data shown in Figure 8B, it can be seen that the 23 amino acid residue E2 signal peptide which forms the hydrophobic carboxyl-terminus of capsid protein, is required for association with mitochondria. Moreover, the observation that a pool of CapCT overlaps with cytochrome c indicates that the carboxyl-terminal region of capsid protein contains information that is sufficient for targeting to mitochondria. Intriguingly, expression of the CapCT construct caused extreme compaction of the mitochondrial network to the perinuclear region, much more so than in cells expressing full-length 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