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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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K594D exhibits altered capacity for membrane penetration.(A) A 3% v/v solution of bovine erythrocytes was incubated with 5.4×1010 ISVPs of rsT3D or the indicated ϕ mutant at 37°C for 1 h. Hemolysis was quantified by determining absorbance of the supernatant at 415 nm. Hemolysis following treatment of an equal number of cells with virion-storage buffer or virion-storage buffer containing 1% TX-100 was considered to be 0 or 100%, respectively. Results are expressed as mean percent hemolysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) L929 cells preincubated with 51Cr-labeled sodium chromate were adsorbed with 105 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h and incubated at 37°C for 4 h following addition of complete medium. The amount of 51Cr released into the medium was determined by liquid scintillation. 51Cr release following treatment of an equal number of cells with virion-storage buffer or by addition of 4% TX-100 to the medium was considered to be 0 or 100%, respectively. Results are expressed as mean percent lysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) HeLa cells starved of cysteine and methionine were adsorbed with 106 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h. Infection was initiated in medium containing 35S-labeled cysteine and methionine in the presence or absence of α-sarcin. Cells were lysed following incubation at 37°C for 1 h. Proteins were precipitated with TCA, and acid-precipitable radioactivity was quantified by scintillation counting. Results are expressed as mean 35S incorporated for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to 35S incorporated in the absence of α-sarcin. (D) CsCl-treated ISVPs of rsT3D or the indicated μ1 mutant were incubated with trypsin at 4°C for the intervals shown. Samples were resolved by SDS-PAGE and immunoblotted using a MAb specific for μ1. The position of the δ band is shown. (E) ISVPs of rsT3D or the indicated μ1 mutant were incubated at the temperatures shown for 15 min. Residual infectivity was assessed by plaque assay. Results are expressed as mean residual titer for triplicate samples. Error bars indicate SD.
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ppat-1000248-g003: K594D exhibits altered capacity for membrane penetration.(A) A 3% v/v solution of bovine erythrocytes was incubated with 5.4×1010 ISVPs of rsT3D or the indicated ϕ mutant at 37°C for 1 h. Hemolysis was quantified by determining absorbance of the supernatant at 415 nm. Hemolysis following treatment of an equal number of cells with virion-storage buffer or virion-storage buffer containing 1% TX-100 was considered to be 0 or 100%, respectively. Results are expressed as mean percent hemolysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) L929 cells preincubated with 51Cr-labeled sodium chromate were adsorbed with 105 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h and incubated at 37°C for 4 h following addition of complete medium. The amount of 51Cr released into the medium was determined by liquid scintillation. 51Cr release following treatment of an equal number of cells with virion-storage buffer or by addition of 4% TX-100 to the medium was considered to be 0 or 100%, respectively. Results are expressed as mean percent lysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) HeLa cells starved of cysteine and methionine were adsorbed with 106 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h. Infection was initiated in medium containing 35S-labeled cysteine and methionine in the presence or absence of α-sarcin. Cells were lysed following incubation at 37°C for 1 h. Proteins were precipitated with TCA, and acid-precipitable radioactivity was quantified by scintillation counting. Results are expressed as mean 35S incorporated for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to 35S incorporated in the absence of α-sarcin. (D) CsCl-treated ISVPs of rsT3D or the indicated μ1 mutant were incubated with trypsin at 4°C for the intervals shown. Samples were resolved by SDS-PAGE and immunoblotted using a MAb specific for μ1. The position of the δ band is shown. (E) ISVPs of rsT3D or the indicated μ1 mutant were incubated at the temperatures shown for 15 min. Residual infectivity was assessed by plaque assay. Results are expressed as mean residual titer for triplicate samples. Error bars indicate SD.

Mentions: The μ1 ϕ domain incorporates an amphipathic helix that is hypothesized to function during membrane penetration [24],[36]. To determine whether mutations in ϕ alter reovirus membrane-penetration efficiency, we tested the capacity of ISVPs generated from each mutant virus to lyse erythrocytes (Figure 3A). The property of reovirus to perturb erythrocyte membrane integrity and cause hemolysis correlates with endosomal membrane penetration [12],[29],[31]. Incubation of bovine erythrocytes with either rsT3D or I595K resulted in efficient erythrocyte lysis, whereas the K594D mutant failed to elicit significant levels of hemolysis. To determine whether the K594D mutant also is altered in penetration of membranes of reovirus-permissive cells, we tested the capacity of wild-type and ϕ mutant viruses to mediate the release of 51Cr from preloaded L929 cells (Figure 3B). Incubation of L929 cells with either rsT3D or I595K resulted in significantly greater 51Cr release than did K594D. These data suggest an important role for ϕ residue Lys594 in membrane penetration.


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)

K594D exhibits altered capacity for membrane penetration.(A) A 3% v/v solution of bovine erythrocytes was incubated with 5.4×1010 ISVPs of rsT3D or the indicated ϕ mutant at 37°C for 1 h. Hemolysis was quantified by determining absorbance of the supernatant at 415 nm. Hemolysis following treatment of an equal number of cells with virion-storage buffer or virion-storage buffer containing 1% TX-100 was considered to be 0 or 100%, respectively. Results are expressed as mean percent hemolysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) L929 cells preincubated with 51Cr-labeled sodium chromate were adsorbed with 105 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h and incubated at 37°C for 4 h following addition of complete medium. The amount of 51Cr released into the medium was determined by liquid scintillation. 51Cr release following treatment of an equal number of cells with virion-storage buffer or by addition of 4% TX-100 to the medium was considered to be 0 or 100%, respectively. Results are expressed as mean percent lysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) HeLa cells starved of cysteine and methionine were adsorbed with 106 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h. Infection was initiated in medium containing 35S-labeled cysteine and methionine in the presence or absence of α-sarcin. Cells were lysed following incubation at 37°C for 1 h. Proteins were precipitated with TCA, and acid-precipitable radioactivity was quantified by scintillation counting. Results are expressed as mean 35S incorporated for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to 35S incorporated in the absence of α-sarcin. (D) CsCl-treated ISVPs of rsT3D or the indicated μ1 mutant were incubated with trypsin at 4°C for the intervals shown. Samples were resolved by SDS-PAGE and immunoblotted using a MAb specific for μ1. The position of the δ band is shown. (E) ISVPs of rsT3D or the indicated μ1 mutant were incubated at the temperatures shown for 15 min. Residual infectivity was assessed by plaque assay. Results are expressed as mean residual titer for triplicate samples. Error bars indicate SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600812&req=5

ppat-1000248-g003: K594D exhibits altered capacity for membrane penetration.(A) A 3% v/v solution of bovine erythrocytes was incubated with 5.4×1010 ISVPs of rsT3D or the indicated ϕ mutant at 37°C for 1 h. Hemolysis was quantified by determining absorbance of the supernatant at 415 nm. Hemolysis following treatment of an equal number of cells with virion-storage buffer or virion-storage buffer containing 1% TX-100 was considered to be 0 or 100%, respectively. Results are expressed as mean percent hemolysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) L929 cells preincubated with 51Cr-labeled sodium chromate were adsorbed with 105 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h and incubated at 37°C for 4 h following addition of complete medium. The amount of 51Cr released into the medium was determined by liquid scintillation. 51Cr release following treatment of an equal number of cells with virion-storage buffer or by addition of 4% TX-100 to the medium was considered to be 0 or 100%, respectively. Results are expressed as mean percent lysis for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) HeLa cells starved of cysteine and methionine were adsorbed with 106 ISVPs/cell of rsT3D or the indicated ϕ mutant at 4°C for 1 h. Infection was initiated in medium containing 35S-labeled cysteine and methionine in the presence or absence of α-sarcin. Cells were lysed following incubation at 37°C for 1 h. Proteins were precipitated with TCA, and acid-precipitable radioactivity was quantified by scintillation counting. Results are expressed as mean 35S incorporated for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to 35S incorporated in the absence of α-sarcin. (D) CsCl-treated ISVPs of rsT3D or the indicated μ1 mutant were incubated with trypsin at 4°C for the intervals shown. Samples were resolved by SDS-PAGE and immunoblotted using a MAb specific for μ1. The position of the δ band is shown. (E) ISVPs of rsT3D or the indicated μ1 mutant were incubated at the temperatures shown for 15 min. Residual infectivity was assessed by plaque assay. Results are expressed as mean residual titer for triplicate samples. Error bars indicate SD.
Mentions: The μ1 ϕ domain incorporates an amphipathic helix that is hypothesized to function during membrane penetration [24],[36]. To determine whether mutations in ϕ alter reovirus membrane-penetration efficiency, we tested the capacity of ISVPs generated from each mutant virus to lyse erythrocytes (Figure 3A). The property of reovirus to perturb erythrocyte membrane integrity and cause hemolysis correlates with endosomal membrane penetration [12],[29],[31]. Incubation of bovine erythrocytes with either rsT3D or I595K resulted in efficient erythrocyte lysis, whereas the K594D mutant failed to elicit significant levels of hemolysis. To determine whether the K594D mutant also is altered in penetration of membranes of reovirus-permissive cells, we tested the capacity of wild-type and ϕ mutant viruses to mediate the release of 51Cr from preloaded L929 cells (Figure 3B). Incubation of L929 cells with either rsT3D or I595K resulted in significantly greater 51Cr release than did K594D. These data suggest an important role for ϕ residue Lys594 in membrane penetration.

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