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Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

Danthi P, Coffey CM, Parker JS, Abel TW, Dermody TS - PLoS Pathog. (2008)

Bottom Line: We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates.Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery.These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, USA. pdanthi@indiana.edu

ABSTRACT
Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

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ϕ mutant viruses are apoptosis-defective in vivo.ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated ϕ mutant. Brains from infected mice were resected at the times shown and homogenized by freeze-thaw and sonication. (A) Brain homogenates were resolved by SDS-PAGE and immunoblotted using an antiserum specific for activated caspase-3 (C-3) (upper panel). As a control for protein concentration, the blots were stripped and reprobed with an antibody specific for actin (lower panel). (B) Brain homogenates from five animals infected with each virus strain for the times shown were resolved by SDS-PAGE and immunoblotted using antisera specific for either activated caspase-3 or actin. Pixel intensities of activated caspase-3 and actin bands were quantified by densitometry. Results are expressed as the mean ratio of activated caspase-3 band intensity to that of actin. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point. (C) For each brain homogenate, the cleaved caspase-3/actin ratio obtained by immunoblot analysis was divided by the virus titer. Results are expressed as the mean activated caspase-3 concentration/PFU. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point.
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ppat-1000248-g008: ϕ mutant viruses are apoptosis-defective in vivo.ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated ϕ mutant. Brains from infected mice were resected at the times shown and homogenized by freeze-thaw and sonication. (A) Brain homogenates were resolved by SDS-PAGE and immunoblotted using an antiserum specific for activated caspase-3 (C-3) (upper panel). As a control for protein concentration, the blots were stripped and reprobed with an antibody specific for actin (lower panel). (B) Brain homogenates from five animals infected with each virus strain for the times shown were resolved by SDS-PAGE and immunoblotted using antisera specific for either activated caspase-3 or actin. Pixel intensities of activated caspase-3 and actin bands were quantified by densitometry. Results are expressed as the mean ratio of activated caspase-3 band intensity to that of actin. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point. (C) For each brain homogenate, the cleaved caspase-3/actin ratio obtained by immunoblot analysis was divided by the virus titer. Results are expressed as the mean activated caspase-3 concentration/PFU. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point.

Mentions: To quantitatively test whether the ϕ mutant viruses differ from wild-type virus in apoptosis-inducing capacity, we examined brain homogenates at 6, 8, and 10 d post-inoculation for the activated form of caspase-3 by immunoblotting (Figure 8A). Activated caspase-3 levels in the brain homogenates of five randomly selected mice following infection with each virus strain were normalized to the levels of actin (Figure 8B). At each interval tested, brain homogenates from mice infected with rsT3D displayed a substantially higher level of activated caspase-3 in comparison to those infected with either K594D or I595K. Although the mean viral titers in the brains of mice infected with rsT3D, K594D, and I595K are equivalent (Figure 6B), it is possible that differences in the concentration of activated caspase-3 in an individual mouse brain (shown in Figure 7B) are a consequence of differences in viral replication. To control for such differences, the in vivo apoptotic potential of each virus was expressed as a ratio of the concentration of activated caspase-3 per PFU of virus (Figure 8C) for each infected mouse brain. In comparison to rsT3D, we found significantly lower levels of activated caspase-3/PFU following infection with K594D (∼70-fold) and I595K (∼10-fold) at 8 d post infection. Although apoptosis levels increase at 10 d post infection following infection with each virus, the activated caspase-3/PFU ratio remained lower following infection with K594D (∼8-fold) and I595K (∼3-fold) in comparison to rsT3D. These data indicate that the ϕ mutant viruses are defective in apoptosis induction in vivo and provide further evidence that the attenuated virulence of K594D and I595K is attributable to a reduced capacity to evoke apoptotic cell death.


Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

Danthi P, Coffey CM, Parker JS, Abel TW, Dermody TS - PLoS Pathog. (2008)

ϕ mutant viruses are apoptosis-defective in vivo.ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated ϕ mutant. Brains from infected mice were resected at the times shown and homogenized by freeze-thaw and sonication. (A) Brain homogenates were resolved by SDS-PAGE and immunoblotted using an antiserum specific for activated caspase-3 (C-3) (upper panel). As a control for protein concentration, the blots were stripped and reprobed with an antibody specific for actin (lower panel). (B) Brain homogenates from five animals infected with each virus strain for the times shown were resolved by SDS-PAGE and immunoblotted using antisera specific for either activated caspase-3 or actin. Pixel intensities of activated caspase-3 and actin bands were quantified by densitometry. Results are expressed as the mean ratio of activated caspase-3 band intensity to that of actin. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point. (C) For each brain homogenate, the cleaved caspase-3/actin ratio obtained by immunoblot analysis was divided by the virus titer. Results are expressed as the mean activated caspase-3 concentration/PFU. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600812&req=5

ppat-1000248-g008: ϕ mutant viruses are apoptosis-defective in vivo.ND4 Swiss Webster mice were inoculated intracranially with 50 PFU of rsT3D or the indicated ϕ mutant. Brains from infected mice were resected at the times shown and homogenized by freeze-thaw and sonication. (A) Brain homogenates were resolved by SDS-PAGE and immunoblotted using an antiserum specific for activated caspase-3 (C-3) (upper panel). As a control for protein concentration, the blots were stripped and reprobed with an antibody specific for actin (lower panel). (B) Brain homogenates from five animals infected with each virus strain for the times shown were resolved by SDS-PAGE and immunoblotted using antisera specific for either activated caspase-3 or actin. Pixel intensities of activated caspase-3 and actin bands were quantified by densitometry. Results are expressed as the mean ratio of activated caspase-3 band intensity to that of actin. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point. (C) For each brain homogenate, the cleaved caspase-3/actin ratio obtained by immunoblot analysis was divided by the virus titer. Results are expressed as the mean activated caspase-3 concentration/PFU. Error bars indicate SEM. *, P<0.05 as determined by Student's t test in comparison to rsT3D at each time point.
Mentions: To quantitatively test whether the ϕ mutant viruses differ from wild-type virus in apoptosis-inducing capacity, we examined brain homogenates at 6, 8, and 10 d post-inoculation for the activated form of caspase-3 by immunoblotting (Figure 8A). Activated caspase-3 levels in the brain homogenates of five randomly selected mice following infection with each virus strain were normalized to the levels of actin (Figure 8B). At each interval tested, brain homogenates from mice infected with rsT3D displayed a substantially higher level of activated caspase-3 in comparison to those infected with either K594D or I595K. Although the mean viral titers in the brains of mice infected with rsT3D, K594D, and I595K are equivalent (Figure 6B), it is possible that differences in the concentration of activated caspase-3 in an individual mouse brain (shown in Figure 7B) are a consequence of differences in viral replication. To control for such differences, the in vivo apoptotic potential of each virus was expressed as a ratio of the concentration of activated caspase-3 per PFU of virus (Figure 8C) for each infected mouse brain. In comparison to rsT3D, we found significantly lower levels of activated caspase-3/PFU following infection with K594D (∼70-fold) and I595K (∼10-fold) at 8 d post infection. Although apoptosis levels increase at 10 d post infection following infection with each virus, the activated caspase-3/PFU ratio remained lower following infection with K594D (∼8-fold) and I595K (∼3-fold) in comparison to rsT3D. These data indicate that the ϕ mutant viruses are defective in apoptosis induction in vivo and provide further evidence that the attenuated virulence of K594D and I595K is attributable to a reduced capacity to evoke apoptotic cell death.

Bottom Line: We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates.Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery.These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, USA. pdanthi@indiana.edu

ABSTRACT
Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Show MeSH
Related in: MedlinePlus